起動する前に1.提言<ol><li>彼らは洞結節細胞に適切に区別しないので、マイコプラズマで汚染されたのPSCを使用しないでください。プロトコルを開始する前に、マイコプラズマ汚染のためのテスト。マイコプラズマの迅速、高感度検出のためのPCRキットを使用してこれを行うとmanufacturer`sプロトコルに従う。 </li><li>各ペトリ皿（ステップ2.3.4）、被膜37°Cで1時間冷水魚皮から無菌7ミリリットル0.1％ゼラチンで1 10cm 2の細胞培養皿である。ゼラチンを取り外し、無菌ベンチで滅菌条件下で食器を乾燥させる。 </li><li>哺乳動物発現ベクターを用いてTBX3のI）構成的過剰発現：あなたが分化プロトコルを開始する前に、次の機能を含む二トランスフェクションし、安定したマウスES細胞株クローンを必要としています。 ⅱ）MYH6-プロモーター5の制御下のG418耐性遺伝子。 </li><li>未分化のPSC細胞を共育成されるべきである記載1のように、標準的な条件下で照射されたフィーダー細胞とdは。 </li></ol> 2.分化手順<ol><li>二日分化する前に - フィーダー細胞からのPSCを削除します。 <ol><li> 10mlのリン酸緩衝生理食塩水PBSで細胞を洗浄し、培地を吸引7ミリリットルのコラゲナーゼIV溶液を添加し、37℃で10分間細胞をインキュベートする。 50ミリリットルチューブに無菌の40μmのフィルターを配置します。 </li><li>注意深くPSCコロニーをすすぎ上下に5回のコラゲナーゼ溶液をピペッティングすることによって（フィーダー細胞を除去することを避ける）。 </li><li> 8ミリリットルのPBSでフィルターを3回すすぎ、40μmのフィルターに細胞懸濁液を転送します。フィルターの周りに回して、ペトリ皿に逆さまに置きます。フィルターの底に10ミリリットルのPBSをピペットによりPSC細胞コロニーを削除します。 </li><li> 300×gで5分間、15mlチューブと遠心分離機に細胞懸濁液を移す。 </li><li> PBSを削除し、1ミリリットルのAccutaseで細胞を懸濁し、I5分間37℃でncubate。 </li><li> 10mlのPBSを追加し、300×gでダウンして5回5分間遠心分離し、それをピペッティングして細胞溶液を混ぜる。 </li><li> 、PBSを削除10ミリリットル培養培地中で細胞を懸濁し、細胞数を決定する。 </li><li>シード75cm 2の濾過フラスコに15000細胞/ cm 2、37℃、5％CO 2で2日間それらを養う。 2日後、フラスコを50〜70％コンフルエントにする必要があります。 </li></ol></li><li> 0日目 - 分化のスタート<ol><li>培地を除去し、10mlのPBSで細胞を洗浄。 </li><li> PBSを吸引し、2ミリリットルのAccutaseを追加し、5分間37℃で細胞をインキュベートする。 50ミリリットルチューブに無菌の40μmのフィルターを配置します。 </li><li> 、40μmのフィルターに細胞懸濁液を転送、10mlのPBSを追加10mlのPBSを添加して遠心分離を介してフィルタをすすぎ、フロースルー300×gで5分間。 </li><li> 10ミリリットルの分化培地中で細胞を懸濁し、細胞数を決定する。 </li><li>トンを希釈彼は20,000細胞/ mlの最終濃度に分化培地を有する懸濁液を細胞。 </li><li>ピペット20mlの水とのHDの乾燥アウトを回避するために次のペトリ皿の低下（HD）溶液をぶら下げ5ミリリットル。 注：iSABsを含む各24ウェルプレートの場合（セクション2.8.6.4/2.8.7を参照）16ペトリ皿で​​始まる。 </li><li>トレイ50mlの細胞懸濁液までピペット。 </li><li>ペトリ皿の蓋の周りに回します。 12チャンネルピペットを用いて、蓋の上に180のHDをそれぞれ含む20μL（400セル/ HD）細胞懸濁液を置きます。 </li><li>慎重に蓋の周りにして、ペトリ皿の上に置きます。 注：蓋の周りに回転の速度が非常に重要です。蓋が遅すぎるか早すぎる周りになっている場合は、細胞懸濁液の表面張力が吊り蓋の上にドロップ保持するのに十分に高くない。メディアの20μlの滴で蓋の周りに回し、初めての練習のためのHDを生産しようとする前に。 </li><li> 3で2日間細胞を培養7℃、5％CO 2は、それらが胚様を形成させた。 注：5ペトリ皿のスタックの上に水を充填した場所ペトリ皿1。そうでない場合は、上ペトリ皿にHDは乾燥します。 </li></ol></li><li> 2日目<ol><li>注意深く次ペトリ皿の蓋の周りにして、50mlチューブ2つのペトリ皿（360のEB）に由来のEBを転送する。 </li><li> EBを（EBSを遠心しないでください）​​、チューブの底に沈降させるために10分を待ちます。 </li><li>できるだけ媒体として吸引し、10ミリリットルの分化培地中のEBを懸濁さ10cmペトリ皿に懸濁液を移す。 </li></ol></li><li>デイ2-6浮遊培養<ol><li> 37℃、5％CO 2で4日間懸濁液中のEBを養う、2日後に培地を変える。 NOTE：EBはしばしば表面に付着（細胞培養皿ではなく）シャーレに塗布されていないにもかかわらず。毎日添付EBSのペトリ皿を制御し、必要に応じてのEBを切り離す穏やかにピペッティングすることによって表面から。オプション：浮遊培養期間中に連続してペトリ皿を振る。シェーカーの速さには注意してください。 EBは、ペトリ皿の国境に真ん中でもフロート内に蓄積しないはずもない。 </li></ol></li><li> 6日目<ol><li> 1ゼラチンコーティングされた10cm 2の細胞培養皿に1ペトリ皿からのEBを転送します。 </li></ol></li><li>デイ7-12 <ol><li>細胞は8〜12日後に打つために開始する必要があります。毎日顕微鏡下で細胞の増殖巣を破ってないか確認してください。細胞は、層を形成し始める。 </li><li>毎日培地交換。 注：メディアを変更した後、細胞が回復すると再び暴行を開始するために3-4時間を必要とする。 </li></ol></li><li>洞結節細胞のデイ12-15選択。 <ol><li>細胞は（日12-15前後）打ち​​始めた三日後に、培地をピペットの細胞に400 / mlのG418を含有する新鮮な分化培地10mlを吸引。 </li></ol></li><li&gt;は4日の選択の開始後に細胞層を解離。 注：細胞層がすでに表面から剥離されることが可能である。 <ol><li>細胞層を吸引することなく、慎重に培地を除去します。 </li><li> 10mlのPBSを追加し、細胞層を吸引することなく、慎重にPBSを削除します。 </li><li> 50mlチューブに細胞を移し、5分間、37℃で細胞をインキュベートし、コラゲナーゼIV溶液10mlを加える。 </li><li>激しくピペッティングにより10倍ダウンサスをミックスし、5分間、37℃で細胞をインキュベートする。小さなクラスターの懸濁液が発生する必要があります。左層の巨​​大な部分が残っている場合、ステップ2.8.4さらに2回繰り返す。 </li><li> 300×gで10分間、PBS 20 mlの遠心分離を追加する。 </li><li> PBSを慎重に吸引除去する。 注：iSABsが生成されるようになっている場合、ステップ2.8.7に進みます。 ISAB派生洞結節単一細胞が生成される場合には、ステップ2.9に進んでください。 </li><li>細胞を懸濁12mlの分化培地は、400 / mlのG418を含有するコ​​ーティングされたゼラチンの6ウェル24ウェル（/ウェル2ml）中にそれらをシード。 </li></ol></li><li>単一リンパ節​​細胞の生成。 <ol><li> 5ミリリットルのAccutaseのステップ2.8.6から細胞を一時停止し、5分間37℃でインキュベートする。 </li><li>激しくピペッティングにより10倍ダウンサスをミックスし、5分間、37℃で細胞をインキュベートする。 </li><li> 300×gで5分間、20 mlのPBSと遠心を追加します。 </li><li> 400 / mlのG418を含む12ミリリットル分化培地中の細胞を中断し、24ウェル（2ミリリットル/ウェル）コーティングされたゼラチンの6ウェル上にそれらをシード。 </li></ol></li><li>解離後の2日目に、培地を吸引し、PBSで細胞を3回洗浄する。ウェル/ 1ミリリットル分化培地を追加します。 </li><li>デイ4-8解離した後に、すべての第2 回日中に変更します。解離用iSABsまたはISAB派生洞結節の単セルの後10日目にさらなる分析のために利用可能です。 </li></ol>

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著者らは、開示することは何もない。

細胞由来の心臓ペースメーカー幹細胞生産能」とは、生物学的ペースメーカー」の意味での適切な心臓リズムの再構成を可能にすることができる。同様に、in vitroでの薬物検査は、それらの利用の恩恵を受ける。 PSCがペースメーカー細胞特性8,9,10,11,12,13心筋細胞を含む哺乳動物の体の任意の細胞型を生じさせることができる。しかし、典型的には、「胚様ボディ」内の細胞集団は、?...

用語「洞不全症候群は、「心臓ペースメーカーシステムの劣化につながる複数の疾患をまとめたもの。それは病的な、症候性洞性徐脈、洞房ブロック、洞停止だけでなく、頻脈、徐脈症候群を含む。これにより、「洞不全症候群」は、多くの場合、虚血性心疾患、心筋症または心筋炎などの一般的な心疾患を伴っている。現在、治療のアプローチは、電気的ペースメーカーの移植に基づいている。しかし、このような感染症やバッテリ障害などのリスクの数と共に進む。全体として、合併症の発生率は、人工ペースメーカーを移植した患者では依然として非常に高い。内因性のペースメーカーとは対照的に、また、これらのデバイスは、ホルモン刺激に応答しない。 将来の選択肢はいるのPSCが役立つことができる「生物学的ペースメーカー」の利用可能性に依存してもよい適切な細胞源とそれはまた、インビトロ薬物試験のために非常に有用であるように。しかし、大きな問題は、胚様体（EB）内の洞結節細胞の非常にまれな外観にある-これは通常、〜0.5％1を超えない。 以前は、それが特定の心筋細胞のサブタイプに向かって「前方プログラミング」は、関連する中胚葉、固有事後1（MesP1）及びNK2転写因子、遺伝子座5（Nkx2.5の）2のような明確な早期の心血管系の転写因子の過剰発現を介して実行可能であることが示された、 3。洞房結節（SAN）の通常のサイズおよび機能のために、Tボックス転写因子TBX3ペースメーカー遺伝子プログラムを開始し、SAN 4の分化を制御することが示されている、非常に重要である。これは機能的ペースメーカー細胞の出現を高めながら、コンテンツがまだ全体cardiomyocytic細胞集団内の約40％を超えなかった。 したがって、追加のMYH6プロモーターベースの抗生物質選択ステップ5は、私たちによって導入された。 in vitroでの栽培にネズミの心臓のものと同等のを近似初めてin vitroで非常に増加鼓動の周波数（&gt; 400 BPM）を示す;これは最終的にまだ未観測心筋細胞凝集体（iSABs「「誘導された洞房体」）につながるマウスの心臓6から分離洞結節細胞。イソプレナリン政権下では550 BPMのさえ破っ周波数が達成される。注目すべきは、iSABsは7を解析して大規模な生理的から明らかなように、80％以上の機能的な結節細胞で構成されています。最近では、直接リプログラミング19を使用して、洞結節細胞を生成するには、いくつかのアプローチが、表面が16,17が記述された小分子との14または薬理学的治療マーカー。しかし、これらの方法はいずれも、ペースメーカー細胞のような高純度につながっていないと、マウスに近い周波数を破って、彼iSABsで観察されたように芸術。 さらに、それらの自発的な拍動活性を失った栽培成体マウス脳切片のex vivoモデルにおいて、iSABsそれによって自発的にアクティブ残りとロバスト収縮7に心臓切片ペーシング、スライス組織に組み込むことができる。これらのiSABsを生成するための詳細なプロトコルは、この論文に記載されている。

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			<td height="20" style="height:20px;">Iscove&#39;s liquid medium with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0465</td>
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			<td height="20" style="height:20px;">DMEM liquid medium without Na-pyruvate, with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0435</td>
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			<td>Biochrom AG</td>
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			<td>Biochrom AG</td>
			<td>S 0615</td>
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			<td>Biochrom AG</td>
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			<td>Biochrom AG</td>
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			<td>PAN-Biotech</td>
			<td>P04-36500</td>
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			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>PAN-Biotech</td>
			<td>P30-3302</td>
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			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
			<td>LIF-250</td>
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			<td height="20" style="height:20px;">Quadratic petri dishes</td>
			<td>Roth</td>
			<td>PX67.1</td>
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			<td height="20" style="height:20px;">Gelatin from cold water fish skin</td>
			<td>SigmaAldrich</td>
			<td>G7765</td>
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			<td height="20" style="height:20px;">2-Mercaptoethanol</td>
			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
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			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
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			<td height="20" style="height:20px;">Tissue culture dishes</td>
			<td>TPP</td>
			<td>93100</td>
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			<td height="20" style="height:20px;">Tissue culture flask</td>
			<td>TPP</td>
			<td>90076</td>
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			<td>TPP</td>
			<td>92424</td>
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generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

「洞不全症候群」の治療は、人工ペースメーカーに基づいています。これらには、バッテリ障害や感染症などの危険を負担する。さらに、それらは、ホルモン応答性を欠いており、全体的な手順は、コスト集約的である。のPSCから生成された「生物学的ペースメーカーは、「代替になることがあり、まだ胚様体（EB）におけるペースメーカー細胞の典型的な含有量は極めて低い。記載されているプロトコルはMYH6プロモーターベースの抗生物質選択で洞結節デューサTBX3 を経由して 、ネズミのPSCの「前方プログラミング」を兼ね備えています。これは&gt; 80％の生理学的に機能的なペースメーカー細胞の一貫性の心筋細胞凝集体が得られます。これらの「誘導された-洞房-体」（「iSABs」）自発的にマウス心臓から単離したリンパ節の細胞に対応するまだ未達の周波数（400〜500 BPM）に委託し、ex vivoでのマウス心筋をペーシングすることができますされている。記載されているプロトコル高純度洞の使い方結節単一細胞は、in vitro薬物試験のために使用することができる例を生成することができる。さらに、このプロトコルに従って生成iSABsは、心臓組織工学に向けての重要なステップになることがある。

記載されているプロトコルは、マウス心臓の鼓動周波数に近い（映画で示されている）のPSCから約450 BPMの叩解周波数でiSABsの生成を可能にする。 図1に示すようにiSABsの解離（ステップ2.8.8）の後に観察された単一細胞が洞結節（スピンドルおよびクモ細胞）の細胞の典型的な形状を示す。これらの細胞機能に必須であることが知られている高発現タンパク質Connexin45（Cx45）、Connex...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

このプロトコルは、マウス多能性幹細胞（PSC）から機能洞結節組織を生成する方法について説明します。 T-BOX3（TBX3）過剰発現プラス心臓ミオシン-重鎖（MYH6）プロモーター抗生物質選択は、高純度のペースメーカー細胞凝集につながる。これらの「誘導された-洞房-体」（「iSABs」）、80％以上のペースメーカー細胞を含む非常に増加鼓動率を示し、ex vivoで心筋をペーシングすることができます。

MYH6プロモーター - 選択と組み合わせたES細胞のプログラミングに基づいて、マウス心臓ペースメーカー細胞凝集体の生成

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K ビュー.  University of Rostock. このプロトコルは、マウス多能性幹細胞（PSC）から機能洞結節組織を生成する方法について説明します。 T-BOX3（TBX3）過剰発現プラス心臓ミオシン-重鎖（MYH6）プロモーター抗生物質選択は、高純度のペースメーカー細胞凝集につながる。これらの「誘導された-洞房-体」（「iSABs」）、80％以上のペースメーカー細胞を含む非常に増加鼓動率を示し、ex ...

ビデオ: Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

化学物質安全性のスクリーニングおよびシステム生物学データの生成のために発生毒性アッセイベースのヒト多能性幹細胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

発生生物学

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

対称性の破れ、偏光と創発集団行動を表示、マウス胚性幹細胞の凝集物の生成<em&gt;インビトロ</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

ヒト多能性幹細胞の懸濁液ベースの心臓分化のためのマイクロウェル中の凝集体サイズの最適化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

マウス膵臓前駆細胞の in Vitroからスキャフォールド フリー、三次元のインスリンを表現する Pancreatoids の生成

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

微小トリプシン法によるモザイク乳腺オルガノイドの生成

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

ヒトiPSCベースの血液脳バリアチップの生成

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...