ジュリオ・ディ・ミン博士のphaESCラインの誘導、フローサイトメトリー手術のレモ・フライマン博士に感謝します。また、ミシェル・シャフナー氏とトーマス・M・ヘネク氏が胚移植に関する技術支援を行ったことを認めます。この研究は、スイス国立科学財団(助成金31003A_152814/1)によって支援されました。

<ol>
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著者らは開示するものは何もない。

体細胞核移動による哺乳類のクローニング(SCNT)は、1990年代、20年、21年に開拓された。これらの開発は、両生類22で30年前に行われたクローニング研究に続いた。かなりの遅延は、哺乳類における発生学およびゲノム刷り込みの難しさを反映している。哺乳類SCNTの開発は、精子を置換するためのhaESCの適用の基礎であり、このプロトコルで詳述されている。
セル周期同期は、SCNT23の成功の重要な要素です。これは、このプロトコルでのhaESCの注入の場合でもあります。レシピエントにドナーゲノムを導入するには、染色体破損や胚の発達を損なう無数化を避けるために、細胞周期相を一致させる必要があります。セミクローニングは、2つのゲノムと細胞トップラストに互換性を持たねねねなくてはないほど複雑です。以前の報告では、M相逮捕アンドロジェネティックhaESCの注入は、卵母細胞へのG1相haESCの注入よりも半クローン胚のより良い発達率をもたらしたことを実証した7。このレポートは、セミクローニングに適した同期点としてM-phaseを提案します。したがって、ファエスクは、デメコルシンとのメタフェーズでマイトサイト的に逮捕され、MII卵母細胞の卵母斑に注入され、これは自然にマイオシスのメタフェーズIIで逮捕された。重要なことに、M相停止は、ドナーとレシピエント細胞サイクル間の優れた同期を提供し、高効率のマウスESCで達成することができます。
有糸分裂の間、核膜は分解し、複製された染色体が付着する紡錘の形をする。M相DKO-phaESCを注入した後、姉妹クロマチは擬似極体とザイゴテ7 に分離される(図3E)。その結果、DKO-phaESCからの染色体の単一のセットは、半クローン胚に寄与する。DKO-phaESC染色体の姉妹染色体は、注射後に正しく分離できることが重要です。無傷のDKO-phaESCの原形質膜は、疑似極性体への分離を防ぎます。DKO-phaESCの原形質膜が破裂せず、胚が注射後に卵母質に無傷のDKO-phesCを示した稀な症例を実際に観察した(図3D)。したがって、ピペット処理によってDKO-phaESCの原形質膜を除去するために注意する必要があります。注射中に、卵母細胞の大腸スピンドルの破壊を避けることも同様に重要であり、染色体分離欠陥を引き起こし、異数化を誘発する可能性もある。
哺乳類では、ゲノムインプリンティングは、精子置換としてのファセスクの適用を制限する。パルテノジェネティックhaESCはゲノムインプリントの母体構成を有し、精子は父親の構成を有する。したがって、精子置換として野生型ファセスクを注射した後、完全な期間の子犬の生成は起こっていない。この制限を克服するために、IG-およびH19-DDRの欠失はファエックで設計される。母体Igf2-H19およびGtl2-Dlk1 lociにおけるインプリント発現の修飾は、転写された2細胞胚に基づいて、5.1%以上の頻度を有する半クローンマウスの生成を可能にするゲノムインプリントの構成を変更するのに十分である。これらの観察は、2つのインプリントされた遺伝子を標的とすることで、phaESCのゲノムをマウスの精子に置き換えることができる機能的な父方構成に切り替えることを示唆している。それにもかかわらず、この戦略にはphaESCの恒久的な遺伝子組み換えが必要です。代替戦略として、アンドロジェネティックhaESCを検討することができます。アンドロジェネティックなHAESCは、精子ゲノムに由来し、父方のインプリントを所有しています。野生型アンドロジェネティックhaESCが精子置換術として寄与し、移された胚4、7、24の1.3%から1.9%の頻度で全期の子犬を生成したという報告がある。フルタームの子犬はまた、IGの欠失を伴うアンドロジェネティックなhaESCを注入することによって得られた - とH19-DDRは、移された2細胞胚の20.2%の頻度で24。改変アンドロジェネティックhaESCを用いたセミクローニングの効率の向上は、インプリントが培養中に不安定になる可能性があるためである可能性が高い。刻印欠陥は、重要なDDRの遺伝的欠失によって修正される。
卵母細胞や精子のゲノムに遺伝子組み換えを直接導入するのが難しいので、haESCは親のゲノムを別々に操作するための貴重なツールです。精子の代用としてhaESCを使用すると、マウス生殖細胞系列におけるゲノム編集に顕著な利点をもたらします。最近の研究では、CRISPR/Cas9ベースのゲノム編集と、胚発生に不可欠なインプリンティング領域の特性評価のためのhaESCの応用を組み合わせたものであった12。本研究では、母性マウスの開発におけるH19-およびIG-DMRとの組み合わせにおけるRasgrf1-DMRの役割と、卵子子の発達における7つの異なるDMRの機能を分析した。精子に対するhaESCの置換方法は、原始胚芽細胞の発達におけるDND1タンパク質内の主要アミノ酸を同定し、骨発達24、25、26における遺伝子を同定するための遺伝子スクリーニングアプローチの基礎を形成した。胚発生の重要な要因を特定するためのゲノムインプリンティングと遺伝子スクリーニングに関する研究は、haESCをガメティックゲノムとして適用するためのかなりのアプローチです。

哺乳類では、次世代に遺伝情報を伝達する唯一の細胞である。卵母細胞と精子の融合は、成虫動物に発達するジプロイドジゴテを形成する。ガメティックゲノムの変異は、それによって子孫によって継承され、種1の遺伝的変異を駆動する。生殖細胞系列の変異の導入は、遺伝子機能の特徴付けや疾患モデリングを含む多様な生物学的研究のための遺伝子組換え動物を生産するために適用されている。卵母細胞と精子の両方が末端分化され、増殖を停止した高度に特殊化された細胞です。そのため、ディステットの直接改変は技術的に難しく、特殊なアプローチが開発されている。遺伝子組み換えは、遺伝子組み換えESCを胚盤胞に注入することによってマウス生殖細胞系列に導入され、そこで発達中の胚に統合され、生殖細胞系列を植民地化する。また、CRISPR/Cas9システムを含むゲノム編集手法を用いた接合体の遺伝子改変が広く採用されている。
近年、ゲーミティックゲノム3、4、5、6、7、8の代替としてhaESCを適用する顕著なアプローチが報告されている。HaESCsは、パルテノジェネティックまたはアンドロジェネティックハプロイド胚盤胞の内側の細胞塊に由来する幹細胞株であり、染色体4、7、9、10の単一のセットを有する。また、検体ゲノムとアンドロジェネティックhaESCの両方が、卵母細胞への細胞質内注射後に半クローンマウスのゲノムに寄与することが実証されている。他のアプローチとは対照的に、haESCのゲノムは自己再生能力のために培養中に直接改変することができる。精子をhaESCsに置き換えることによって生殖細胞系列に遺伝子組換えを導入することは、生物学的研究にとって重要な方法です。これは、それぞれパルテノジェネティックまたはアンドロジェネティックhaESCに由来する母体または父方のゲノムを分離して培養し、操作する可能性を提供します。その後、HaESCは、ゲノムインプリンティング、アレーレ特異的発現、および親固有のプロセスの研究に特に有利な、ガメティックゲノム置換として使用することができます。
マウスでは、正常胚発生11には母体と父方のゲノム情報の両方が必要である。したがって、精子ゲノム5,8を置換するために野生型パルテノジェネティックhaESC(phaESC)を注入した場合、完全な子犬は得ることができなかった。この発達ブロックを克服するためには、パルテノジェネティックhaESCの母体ゲノムのゲノム刷り込みは、父方の構成に修正する必要があります。これは、微分メチル化領域(DDR)の操作によって達成することができる。現在までに、H19-DMR、Gtl2-Dlk1 IG-DMR、およびRasgrf1-DMRの標的欠失が、phaESC3、5、8、12において母体的に発現した遺伝子を抑制することを研究されてきた。 これらの研究は、H19-DMRとIG-DMRの両方の欠失は、精子染色体の代わりにすることができる父方のインプリント構成に母親を変換するのに十分であることを実証した。卵母細胞に2つのDMR欠失を運ぶphaESCの細胞内皮注入は、移された2細胞胚の5.1%から15.5%の頻度を有する半クローンの子犬を生み出した。
このプロトコルは 、H19-DMR と IG-DMR の両方を削除した phaESC の適用に基づいています。我々は、DKO-phaESCラインを確立するためのファエスクにおけるゲノムインプリンティングの改変、精子ゲノムの代替として卵母細胞へのDKO-phesCの注入、半クローン胚の培養、半クローンマウスの取得に関する詳細な指示を提供する。このプロトコルは、父親のゲノムの正確かつ直接的な操作と半クローン胚およびマウスの生成を必要とする研究者のための参照である。

<table>
	<tbody>
		<tr>
			<td>1.5 mLTube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>15 ml Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Thermo Scientific</td>
			<td>150628</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Merck</td>
			<td>M6250</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>BSA fraction</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Axon</td>
			<td>1386</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Demecolcine solution</td>
			<td>Merck</td>
			<td>D1925</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM / F-12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DR4 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>3208</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>E5134</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>biowest</td>
			<td>S1810-500</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td>amsbio</td>
			<td>11897</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Merck</td>
			<td>G1890</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Hepes solution</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Irradiated MEF</td>
			<td>Gibco</td>
			<td>A34966</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>LIF (Leukemia inhibitory factor)</td>
			<td>(Homemade)</td>
			<td>-</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Lipofection reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>NDiff 227</td>
			<td>Takara</td>
			<td>Y40002</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>PD 0325901</td>
			<td>Axon</td>
			<td>1408</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac plasmid carrying a CAG-EGFP transgene</td>
			<td>Addgene</td>
			<td>#40973</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac transposase plasmid</td>
			<td>System Biosciences</td>
			<td>PB210PA-1</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX330 plasmid</td>
			<td>Addgene</td>
			<td>#42230</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX458 plasmid</td>
			<td>Addgene</td>
			<td>#48138</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Thermo Scientific</td>
			<td>F549S</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>100317</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>109634</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Axon</td>
			<td>A3830</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck</td>
			<td>71729</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Epeendorf</td>
			<td>5384000012</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>T1503</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>5 mL Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>5 mL Tube with cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>MoFlo Astrios EQ</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Codan</td>
			<td>62.1640</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>30-gauge 1/2 inch needle</td>
			<td>Merck</td>
			<td>Z192362-100EA</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>B6D2F1 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>100006</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>MSD animal health</td>
			<td>49451</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010015</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Avivasysbio</td>
			<td>OPPA1037 5000 IU</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>10-cm dish</td>
			<td>Thermo Scientific</td>
			<td>150350</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>4-well plate</td>
			<td>Thermo Scientific</td>
			<td>179830</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>6 cm dish</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Center-well dish</td>
			<td>Corning</td>
			<td>3260</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Digital rocker</td>
			<td>Thermo Scientific</td>
			<td>88880020</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>AppliChem</td>
			<td>A0878</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Merck</td>
			<td>H4272</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-5F</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M16 medium</td>
			<td>Merck</td>
			<td>M7292</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Merck</td>
			<td>M7167</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Merck</td>
			<td>M8410</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Merck</td>
			<td>P1049-1PAK</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Strontium chloride</td>
			<td>Merck</td>
			<td>13909</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>5ml syringe</td>
			<td>Codan</td>
			<td>62.5607</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Borosilicate glass cappilary</td>
			<td>Science product</td>
			<td>GB100T-8P</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>CellTram 4r Oil</td>
			<td>Eppendorf</td>
			<td>5196000030</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Fluorinert FC-770</td>
			<td>Merck</td>
			<td>F3556</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Holding pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microinjection pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microlaoder tips</td>
			<td>Eppendorf</td>
			<td>5252 956.003</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micromanipulaor arms</td>
			<td>Narishige</td>
			<td>NT-88-V3</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PiezoXpert</td>
			<td>Eppendorf</td>
			<td>5194000016</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PVP (Polyvinylpyrrolidine)</td>
			<td>Merck</td>
			<td>PVP360</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>SARSTEDT</td>
			<td>83.1826.001</td>
			<td>Microinjection</td>
		</tr>
	</tbody>
</table>

application of mouse parthenogenetic haploid embryonic stem cells as a substitute of sperm

性生殖を伴う生物では、生殖細胞は新しい個体に発達する全能性細胞の源である。マウスでは、精子によって卵母細胞の受精は、トーティポテンスの接合子球を作成します。最近、ハプロイド胚性幹細胞(haESC)がガミスティックゲノムの代用となり、マウスに発達する胚に寄与することが報告されています。ここでは、卵母細胞への細胞質注入によって胚を構築する精子の代替としてパルテノジェネティックhaESCを適用するプロトコルを提示する。このプロトコルは、精子置換としてhaESCを調製するためのステップ、卵母細胞へのhaESC染色体の注入、および半クローン胚の培養のためのステップで構成される。胚は、胚移植後に肥沃な半クローンマウスを生み出すことができる。精子置換にHAESCを用い、生殖細胞のゲノム編集、胚発生の研究、ゲノムインプリンティングの研究が容易になります。

Watch this Scientific Journal Video about Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm at JoVE.com

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

この記事は、半クローン胚の構築のための精子の代わりにパルテノジェネティックハプロイド胚性幹細胞の使用を実証することを目的としています。

マウスパルテノジェネティックハプロイド胚性幹細胞の精子代替としての応用

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

application-mouse-parthenogenetic-haploid-embryonic-stem-cells-as

Research

JoVE Journal

Developmental Biology

4.5K ビュー.  ETH Zurich. この記事は、半クローン胚の構築のための精子の代わりにパルテノジェネティックハプロイド胚性幹細胞の使用を実証することを目的としています。

ビデオ: Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

発生生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

無血清単層培養中のマウス胚性幹細胞の神経分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9媒介1アレル欠失マウス胚性幹細胞でエンハンサー機能を研究するために、

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平全マウント：皮膚の厚い3次元組織断面のための新規処理および画像化プロトコール

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...