この作品は、米国癌協会の研究学者グラント（RSG-12-148-01-DDC）とCIRM基本生物学賞（RB4-05779）supportedbyました。

ヒトの皮膚のプロトコールは、カリフォルニア大学サンディ​​エゴ校の研究倫理委員会のガイドラインに従って行いました。ヒトの皮膚は、廃棄された外科的サンプルから得られたか（スキンバンクは材料/機器表に記載されている）は、皮膚の銀行から購入することができます。皮膚が由来されている場所やドナーの年齢は限り真皮における基底膜領域タンパク質（コラーゲン/ラミニン）が分解されないように、実験には重要ではありません。 失活ヒト真皮の調製<ol><li>ヒトの皮膚を受信すると、（〜3-5分）を解凍し、組織培養フード内で組織を置きます。皮膚のサイズに応じて、滅菌使い捨て50mlコニカルチューブまたは125ミリリットル瓶で解凍組織を配置します。皮膚バンクからの皮膚の一般的なサイズは、0.75平方フィートです。 </li><li> 1×PBSで4倍のペニシリンおよびストレプトマイシン（ペニシリン/ストレプトマイシン）で、75％の完全な容器を満たします。激しく振ります5分とは、液体の処分。このステップを3回繰り返します。 </li><li>最後の洗浄後、新しいチューブ/ボトルに組織を転送し、コンテナの75％を埋めるために、新鮮な4倍ペニシリン/ストレプトマイシン/ PBSを追加します。表皮と真皮の分離を可能にするために2週間、37℃の組織培養インキュベーター中でこの混合物をインキュベートします。 </li><li>無菌条件下で、真皮から表皮を剥がすために鉗子を使用しています。表皮は、ドナー（ 図1A）のレースに応じてカラーリングを持つことになりますしながら、真皮は完全に白です。ヒト組織に対処するための金融機関のプロトコルに従って表皮を廃棄します。 </li><li>チューブや瓶の中に真皮を置き、（4回5分間の洗浄）激しく振盪しながら1×PBSで希釈した4倍ペニシリン/ストレプトマイシンでそれを洗います。最後の洗浄後、使用する準備ができるまで4℃で4倍ペン/ 1×PBSで希釈した連鎖球菌およびストアに新しいチューブ/ボトルに真皮を転送します。真皮は今年まで4℃で保存することができます。 </li></オール&gt; 2.遺伝的に変更初代ヒトケラチノサイト注：初代ヒトケラチノサイトに感染するウイルスを生成するために、完全DMEM培地[DMEM + 10％ウシ胎児血清（FBS）+ペニシリン/ストレプトマイシン]中で増殖させた使用アンホトロピックフェニックス細胞。このようなGAG-pol及びエンベロープのようなタンパク質をコードするウイルスパッケージング遺伝子を安定的に、レトロウイルスベクターでトランスフェクトされた場合、ウイルス産生を可能にするフェニックス細胞ゲノムに組み込まれています。フェニックス細胞は、高いウイルス力価の産生を可能にする、高効率でトランスフェクトすることができます。この包装ラインから産生されたウイルスは、ヒトを含む哺乳類細胞の多種多様に感染することができます。 <ol><li> 1日目：トランスフェクションの前日、完全DMEM培地でも80万個の細胞の密度で6ウェルプレートでシードフェニックス細胞。 </li><li> 2日目：トランスフェクションの日には、ウェル当たりレトロウイルスベクターの3μgのを使用しています。アリコート1.5mlチューブへのレトロウイルスベクターの3μgの。 100μのミックスを使用します;リットルのDMEMを加えたベクトルのすべての3μgのためのトランスフェクション試薬の6μlの。 1.5mlチューブ中の100μlのDMEMでトランスフェクション試薬の6μLを混ぜます。 （使用されているレトロウイルスベクターは、装置/材料の表に記載されています）。 <ol><li> 5分間インキュベートし、レトロウイルスベクターの3μgのを含む事前に小分けチューブに106μlの混合物を追加します。混合し、室温で30分間インキュベートします。フェニックス細胞の各ウェルに、この混合物全体を滴下して加えます。 </li></ol></li><li> 3日目：トランスフェクションの翌日、フェニックス細胞から培地を除去し、新鮮な完全DMEM培地2mlのを追加。同じ日に、ウェルあたり75,000細胞の密度で6ウェルプレートに初代ヒトケラチノサイトをシード。抗生物質（ペニシリン/ストレプトマイシン）でケラチノサイト無血清培地（KCSFM）でケラチノサイトを維持します。 注：一次ヒトケラチノサイトを購入しました。細胞は、（材料/機器表に記載されている）、さまざまなベンダーから購入することができます。 </li><li> 4日目：ハー今ウイルス粒子を含む、トランスフェクトされたフェニックス細胞から培地を権利が確定します。注射器を用いて0.45μmのフィルターを通してメディアを渡します（任意の汚染フェニックス細胞を除去するため）、ケラチノサイトに2ミリリットルを配置（前日/ウェル75,000細胞で播種：ステップ2.3を参照してください）​​。感染過程を媒介するために役立つ培地にヘキサジメトリンブロミド（5μg/ ml）を加えます。ウイルス力価は、約1×10 7 TU / mlです。 <ol><li> RTで1時間、200×gで遠心分離で6ウェルプレートをスピン。スピンした後、ウイル​​スを含む培地を除去し、KCSFMを追加する前に、1×PBSで細胞を1回洗浄します。 </li></ol></li><li> 5日目：ステップ2.4​​のように再びケラチノサイトの同じバッチに感染します。 注：レトロウイルスベクターの選択マーカーがある場合、薬物を使用して、ケラチノサイトを選択して、下流の分析や、器官培養などのアプリケーションで使用するために展開します。 </li></ol> 3.器官皮膚の文化を設定します<ol><li>ビルド一般的な実験で見つかった物質またはカセットから器官カセットは、カスタム金属加工工場（ - F図2A）を介して行うことができます。 3.5センチメートル組織培養プレートからこれらのカセットを作るために3.5センチの皿（ 図2A-B）の蓋の中心から1.0センチメートルX 1.0センチメートルの正方形を削除するには、焼灼を使用しています。無菌条件下でこれを行います。 <ol><li>明確なマニキュア（ 図2C）を使用して、カセット（3.5センチの皿の蓋）の底部に正方形のペグを取り付けます。マニキュアのための5分を乾燥し、それが正方形のペグ（ 図2D）に載っているように、カセットを裏返しすることができます。 6cm皿にカセットを置きます。 注：スクエアペグは、芸術品や工芸品店の様々なから購入することができます。 </li></ol></li><li> 10％FBS、2.67 / mlのアデニン、40 N、ストレプトマイシン100μg/ mlの、66％DMEM、22％ハムF12、100単位/ mlのペニシリンからなるケラチノサイト増殖培地（KGM）を準備しますグラム/ mlのヒドロコルチゾン、10 ngの/ mlのコレラ毒素、4.94 / mlのインスリン、5μg/ mlのトランスフェリン、1.36 ngの/ mlのトリヨード - L - チロニン、10 ngの/ mlのEGF、および10μg/ mlのシプロフロキサシン塩酸塩。使用する準備ができるまで4℃で0.22μmフィルターとストアを介してメディアをフィルタリングします。 </li><li> 4倍ペニシリン/ストレプトマイシン+ PBS溶液から真皮を取り、KGMで2回洗浄した後、使用前に2日間37℃でKGMでインキュベートします。 </li><li> 1.5センチメートルX 1.5センチメートルサイズの部分にメスを用いて真皮をカットして、器官カセットの上に置きます。上を向いて真皮の上部と真皮を置きます。真皮の上部には、ケラチノサイト（ 図1B）と接触する側になります。 </li><li> （ 図3A）を上に向け、真皮の上部側と器官カセットの角穴にプレカット真皮を置きます。 </li><li>鉗子を使用しての上に真皮を含む全体の器官カセットをフリップ（図3B）。氷上で細胞外マトリックス溶液を解凍します。カセットあたりの細胞外マトリックス溶液100μlを引き出すために、針と注射器を使用してください。真皮（光沢のある側）の底部に5滴を追加し、真皮（ 図3B）全体に均一な分布を確実にするために、わずかに振ります。 </li><li>商業細胞外マトリックス溶液で5分間は完全に（ 図3C）を固化することを許可します。固化した後、バックの上にカセットを反転するために鉗子を使用しています。 6cmディッシュにKGMの4ミリリットルを追加します。 </li><li>真皮（ 図3D）を含む器官カセット上に0.5から1000000遺伝子改変されたケラチノサイトを置きます。 <ol><li>トリプシン処理を、5分間、10cmのプレートに0.05％の4 mlのトリプシンを配置することにより、ケラチノサイトおよび完全DMEM培地4mlでクエンチしました。 </li><li>血球計数器を使用して、ケラチノサイトをカウントし、その後5分間200×gでスピンダウン。 90μで（利用可能なセルの数に応じて）0.5から1000000細胞を再懸濁しKGMのLと真皮にすべてのセルを配置します。 注：再生表皮の厚さに差が見られる任意の細胞数の開始の違いによるものではないことを保証するために、同じ実験内の真皮に同数の細胞を配置することが重要です。真皮に0.5未満万個の細胞を配置すると、貧しい階層と差別につながることができます。 </li></ol></li><li>一日おきに器官培養にKGMメディアを変更します。収穫組織真皮のケラチノサイトの配置後1-14日。表皮の完全な層化と分化は、通常は5日後に発生します。 </li></ol>

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C., McGuire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+skin:+fibroblasts+facilitate+keratinocyte+growth+and+differentiation+on+acellular+reticular+dermis.">In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.</a> J Invest Dermatol. 97, 843-848 (1991).</li><li>Mathes, S. H., Ruffner, H., Graf-Hausner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+skin+models+in+drug+development.">The use of skin models in drug development.</a> Adv Drug Deliv Rev. 69-70, 81-102 (2014).</li></ol>

We have nothing to disclose.

ヒト皮膚器官培養における遺伝子操作は、一般的に、2D培養細胞だけでなく、マウスモデルを研究するために多くの利点を提供します。 2D培養物は、無傷の組織および器官に見出される三次元の細胞 - 細胞および細胞 - 細胞外マトリックス相互作用を欠いています。最近の研究では、一次ヒト皮膚腫瘍16にはるかに遺伝子発現の類似性を示す3次元培養細胞を用いた2次元および3次元培?...

ヒト表皮基底膜zone.Theの表皮として知られている細胞外マトリックスは、水分の損失を防ぐために、不浸透性の障壁としてだけでなく、保護するための最初の防衛線として機能するだけでなく、を介して基礎となる真皮に接続重層上皮であります外国と有害物質1から体。表皮の最も深い層である基底層は、表皮2の残りの部分を形成する分化した子孫を生じさせる上皮幹細胞および前駆細胞が含まれています。上皮前駆細胞が分化するように、それらは有棘層3として知られている分化した細胞の第1の層を形成するために上方に移動します。棘層では、細胞は、その後、表皮の分化した層のための物理的ストレスに耐える強度を提供する、ケラチン1および10の発現をオンにします。有棘層細胞は、さらに分化するように、それらはのために、表皮中で上方に移動ケラトヒアリンおよびラメラ顆粒の形成と同様に、原形質膜の下に組み立てられている構造タンパク質によって特徴付けられる顆粒層をメートル。細胞は、分化過程に進むように、ラメラ顆粒が細胞から押し出されているが、原形質膜の下のタンパク質は、クロス脂質豊富な障壁を形成するように互いに連結された角質層4と呼ばれます 。 上皮細胞の増殖および分化への影響の変化を伴う疾患人口の5〜20％。したがって、このプロセスのメカニズムを理解することは非常に重要です。これらの疾患の多くの症状は、細胞-細胞または細胞-マトリックス接触時に偶発的であることから、ヒトの表皮は、3D環境で再構成された器官培養物は、6月10日に作成されています。これらの方法は、典型的には、失活ヒトなどの細胞外マトリックス上に播種し、一次または形質転換ケラチノサイトの使用を含みます真皮、マトリゲル、またはコラーゲン。 上皮細胞の増殖および分化において重要な遺伝子調節機構を理解するために、ケラチノサイトを遺伝的に、2D培養での遺伝子ノックダウンまたは過剰発現するようにレトロウイルスベクターを介して操作され、その後、3次元で再構成することができます。これらの方法は、新生物11-21に表皮幹細胞および前駆細胞の自己再生および分化ならびに進行に関与する遺伝子を特徴づけるために広く使用されています。ここでは、レトロウイルスの使用を介して表皮器官培養物中の遺伝子発現を変化させる方法についての詳細なプロトコルが提供されます。

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

器官培養物は、それらのin vivoでの組織の対応の機能および生理学を模倣する細胞-細胞接触および細胞-マトリックス相互作用のための3D環境の再構成を可能にする重要な。これは、忠実に表皮分化と成層プログラムを再現する器官型皮膚培養が挙げられます。初代ヒト表皮角化細胞は、遺伝子が容易に過剰発現またはノックダウンすることができますレトロウイルスを介して遺伝的に操作可能です。これらの遺伝的に改変されたケラチノサイトは、次に遺伝的経路に衝突上皮細胞増殖、分化、および疾患の進行を研究するための強力なモデルを提供し、皮膚器官培養におけるヒト表皮を再生するために使用することができます。ここで紹介するプロトコルは、失活ヒト真皮を準備するだけでなく、と遺伝的に器官皮膚培養を生成するために、初代ヒトケラチノサイトを操作する方法を説明します。再生した人間の皮膚はdownstrに使用することができますこのような遺伝子発現プロファイリング、免疫染色、及びハイスループットシークエンシングに続いてクロマチン免疫沈降などのEAMアプリケーション。したがって、これらの遺伝的に改変された器官、皮膚培養物の生成は、皮膚恒常性を維持するために重要である遺伝子の決意を可能にします。

器官人間の皮膚を生成する最初のステップは、真皮から表皮を除去することです。 37°Cの4倍でペニシリン/ストレプトマイシン/ PBSの皮膚の2週間のインキュベーションは、表皮（ 図1A）から真皮の分離を可能にする必要があります。表皮と真皮を分離することは困難である場合には、別の週に4倍ペニシリン/ストレプトマイシン/ PBS中で37℃で組織を配置し、ピンセットを用いて再...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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Research

JoVE Journal

Developmental Biology

11.1K ビュー.  UCSD Stem Cell Program, University of California, San Diego. The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. 

ビデオ: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

生後神経発生の研究のための器官型スライス培養

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

発生生物学

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

トランスフェクション、選択、およびAAVS1セーフハーバーへのGFP遺伝子とTALENを標的とする人間の人工多能性幹細胞のコロニーピッキング

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

髄質胸腺上皮細胞の増殖、分化および無差別遺伝子発現を支える3D器官共培養モデル

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

脆弱なメキシコ系アメリカ人の青少年に対する修正トリーア社会ストレステスト

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

卵母細胞のマイクロインジェクションによる遺伝子改変マウスの作製

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

世代、大人の肌からプライマリ ヒト角化培養

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

前庭と聴覚の感覚器官の三次元切片培養

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...