著者はモナヘロルドのサポートのためのPaaquaグラントとGWUルーサーライスフェローシップのサポートのためGWUハーラン·フェローシップを承認したいと思います。この作品は、全米科学財団助成MCB-1121711によってサポートされていました。

1。器具、培地や料理の準備<ol><li>アルミナ研磨フィルムを使用して4鉗子を強めています。チップサイズを監視するために解剖顕微鏡下でこれを行います。先端が処置中に損傷している場合には鉗子1組のバックアップとして機能します。すべての4つの鉗子をオートクレーブ滅菌した容器に保管してください。 </li><li> （; 15のレシピどちらMarcの修正リンガー[MMR]または改変バルトの生理食塩水[MBS]）とフィルター滅菌500ミリリットル0.1Xと1.0X培地のそれぞれを確認します。我々は日常的にMBSを（; 1のKCl、0.7 mMのCaCl 2、1mMののMgSO 4、5mMのHEPES [pHが7.8]、2.5 mMのNaHCO 3を 1X = 88 mMのNaCl）を使用します。ヶ月間の14から18℃で保存する。 </li><li>培養液中の2％アガロース溶液（スクリューキャップのガラスボトルで100ミリリットル1X培地で2グラム電気泳動グレードアガロース）を作る。アガロースを溶解するためにオートクレーブ。これは、月間4℃で保存し、それが次の必要なときにアガロースを液化するために電子レンジにすることができます。 </li><li>アガロースは液体ですが、滅菌24穴培養プレートの各ウェルの底に約0.5ミリリットルを注ぐ。これはプラスチックに付着する外植片を防ぐことができます。 </li><li>アガロースは液体ですが、二から三60ミリメートルペトリ皿の底に約2-3ミリリットルを注ぎ、そして渦巻きは優しく底が覆われていることを確認する。これらは、解剖皿となるとその膜が除去された後、アガロースは、プラスチックに付着胚を防ぎます。 </li><li>アガロースを冷却し、硬化、火炎6 &quot;パスツールピペットの先端を、それがボールに溶けるまで、軽く培養プレートの各ウェルにおけるアガロースの表面に接触させてください。これは、先の浅いうつ病が作成されたとき植が配置されます。 </li><li> 1X無菌培養培地で培養プレートの各ウェルを埋める。 </li><li>アガロースが冷却し、硬化したときに、割球分離を容易にするために希釈された培養液（0.1X MMRまたは0.1X MBS）と解剖皿を埋める。 </lI&gt; </ol> 2。胚の選択<ol><li>受精卵を取得し、標準的なプロトコル15に従ってゼリーコートを除去する。 100ミリメートルペトリ皿に0.5X培地に移す。 </li><li>胚は2細胞期に達すると、最初の分裂溝が別の皿に動物半球（ 図1）で軽く色素沈着面積を二等分したものをソートする。これらは、外植片のドナーになります。外植片を上演する兄弟コントロールとして機能するための手順を通してドナー胚の隣に別の皿に残りの2細胞期胚を保管してください。 </li></ol> 3。外植片の作製<ol><li>解剖皿に5月10日胚を置くのではなく、一度に1つの胚に取り組んでいます。 </li><li>透明卵黄膜が見られるように、最初の胚を置き、それは、胚の動物極の表面上に透明な空間（腔）で区切られています。尖った力を利用して1のサブドミナントの手（1が右利きされている場合は左手）のps、腔上記の卵黄膜を把握する。一方で削った鉗子を使用して、最初の鉗子先端に近い膜を持ち、静か膜剥がれ反対方向に引っ張る。標的細胞は、膜の除去の際に破損していないので、解剖するセルからの距離で初期把握を行います。一つは、胚が平らになるため、膜が除去されたことを伝えることができます。 </li><li>サブドミナント手にピンセットで1隣接セルをつかんで、解剖すべきセルが直接触れないように &quot;ハンドル&quot;としてこのセルを使用しています。一方で鉗子で、優しく希望の割球から離れて残りの隣接セルを引っ張る。同じ運命を共有して正中線細胞は、標的にされている場合は、それらがペアとして一緒に削除することができます。最後に、 &quot;ハンドル&quot;のセルを離れて解剖する。 </li><li>滅菌済みの割球（または割球ペア）、拾う、ガラスパスツールピペット、気泡や過度の吸引を避ける。ピペットは、細胞に損傷を与えることができますが、我々が日常的にこれをしないシャープなエッジを除去するために火を研磨することができる。外植片培養皿のウェル中の培地の表面の下にパスツールピペットの先端を置き、静かに割球を追放。それは、重力によって浅いくぼみにスライドしなければなりません。複数の胚から割球は、同じ単植に結合することができます。 </li><li>一つずつ、解剖皿の残りの胚では、この手順を繰り返します。約10胚の後、解剖皿細胞の残骸で満たされるでしょう。この現象が発生した場合、新鮮な解剖皿に変更します。 </li><li>すべての解剖が行われた後、外植片が浅いくぼみにあるかどうかを確認。しない場合、それらは静かに乾燥させ、70％エタノールと空気中で滅菌された毛のループを使ってうつ病に押し込むことができます。 </li><li>最後の郭清後の約一時間、デブリsurrouを削除滅菌、ガラスパスツールピペットで癒さ植片をnding。 </li><li>兄弟、制御の胚を含むペトリ皿に14から20°Cの隣での培養外植片の板を。兄弟の胚は、割球の外植片の発展段階を示すでしょう。 </li></ol> 4。分析のための収穫植<ol><li>兄弟姉妹が希望発達段階に到達したときに外植片を回収します。細胞の一部が崩壊した場合でも、これらの外植片はかなりうまく生き残る。文化の中で曇りの質量が十分に存在する場合、したがって、健康な植が内に埋設されているかどうかを判断するために毛のループまたは鉗子でそれを探る。 </li><li>ガラスパスツールピペットを用いて培養液を少量の外植片を拾うと、優しく行われるアッセイのための適切な固定剤または溶解バッファにそれらを追放する。 </li></ol>

<ol>
	<li>Dale, L., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+32-cell+stage+of+Xenopus+laevis.">Fate map of the 32-cell stage of Xenopus laevis.</a> Development. 100, 279-295 (1987).</li><li>Masho, R., Kubota, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+fates+of+blastomeres+of+the+eight-cell+stage+Xenopus+embryo.">Developmental fates of blastomeres of the eight-cell stage Xenopus embryo.</a> Dev. Growth, Diff. 30, 347-359 (1988).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+16-cell+stage+Xenopus+embryo.">Fates of the blastomeres of the 16-cell stage Xenopus embryo.</a> Dev. Biol. 119, 560-578 (1987).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell+stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell stage Xenopus embryo.</a> Dev. Biol. 122, 300-319 (1987).</li><li>Moody, S. A., Kline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+fate+during+cleavage+of+frog+(Xenopus+laevis)+blastomeres.">Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.</a> Anat. Embryol. 182, 347-362 (1990).</li><li>Takasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+roles+of+the+presumptive+organizer+region+in+the+32-cell+embryos+in+normal+development+of+Xenopus+laevis.">Fates and roles of the presumptive organizer region in the 32-cell embryos in normal development of Xenopus laevis.</a> Dev. Growth, Diff. 29, 141-152 (1987).</li><li>Sullivan, S. A., Moore, K. B., Moody, S. A., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20+Early+events+in+frog+blastomere+fate+determination.">Chapter 20 Early events in frog blastomere fate determination.</a> Cell Fate and Lineage Determination. , 297-321 (1999).</li><li>Moore, K. B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal-vegetal+asymmetries+influence+the+earliest+steps+in+retinal+fate+commitment+in+Xenopus.">Animal-vegetal asymmetries influence the earliest steps in retinal fate commitment in Xenopus.</a> Dev. Biol. 212, 25-41 (1999).</li><li>Yan, B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competence+of+Xenopus+blastomeres+to+produce+neural+and+retinal+progeny+is+repressed+by+two+endo-mesoderm+promoting+pathways.">The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways.</a> Dev. Biol. 305, 103-119 (2007).</li><li>Gallagher, B. C., Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+differentiation+of+dorsal+axial+structures+from+an+animal+cap+cleavage+stage+blastomere+in+Xenopus.">Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus.</a> Development. 112, 1103-1114 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+maternal+mRNAs+from+a+dorsal+animal+blastomere+induce+a+secondary+axis+in+host+embryos.">Xenopus maternal mRNAs from a dorsal animal blastomere induce a secondary axis in host embryos.</a> Development. 116, 347-3345 (1992).</li><li>Godsave, S. F., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+mesoderm+formation+in+the+Xenopus+embryo.">Single cell analysis of mesoderm formation in the Xenopus embryo.</a> Development. 111, 523-530 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activin-like+signal+activates+a+dorsal-specifying+RNA+between+the+8-+and+16-cell+stages+of+Xenopus.">An activin-like signal activates a dorsal-specifying RNA between the 8- and 16-cell stages of Xenopus.</a> Dev. Genetics. 19, 210-221 (1996).</li><li>Pandur, P. D., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+maternal+influences+on+dorsal-ventral+fate+of+Xenopus+animal+blastomeres.">Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.</a> Dev. Dyn. 225, 581-587 (2002).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Development+of+Xenopus+laevis.">Early Development of Xenopus laevis.</a> A Laboratory Manual. , (2000).</li><li>Sullivan, S. A., Akers, L., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=foxD5a,+a+Xenopus+winged+helix+gene,+maintains+an+immature+neural+ectoderm+via+transcriptional+repression+that+is+dependent+upon+the+C-terminal+domain.">foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent upon the C-terminal domain.</a> Dev. Biol. 232, 439-457 (2001).</li><li>Yan, B., Neilson, K. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxD5+plays+a+critical+upstream+role+in+regulating+neural+fate+and+onset+of+differentiation.">FoxD5 plays a critical upstream role in regulating neural fate and onset of differentiation.</a> Dev. Biol. 329, 80-95 (2009).</li><li>Vincent, J. -. P., Gerhart, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcortical+rotation+in+Xenopus+eggs:+an+early+step+in+embryonic+axis+specification.">Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification.</a> Dev. Biol. 123, 526-539 (1987).</li><li>Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+A:+Solutions+and+Protocols.">Appendix A: Solutions and Protocols.</a> Methods in Cell Biology. 36, 657-662 (1991).</li><li>Klein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+cleavage+furrow+demarcates+the+dorsal-ventral+axis+in+Xenopus+embryos.">The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos.</a> Dev. Biol. 120, 299-304 (1987).</li><li>Masho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+the+first+cleavage+plane+and+the+body+axis+in+early+Xenopus+embryos.">Close correlation between the first cleavage plane and the body axis in early Xenopus embryos.</a> Dev. Growth Diff. 32, 57-64 (1990).</li></ol>

特別な利害関係は宣言されません。

ほとんどの個々の割球の培養に成功するための重要なステップは次のとおりである：1）正常に関心の割球を識別する、2）滅菌した、健全な文化を維持すること、3）細胞周期の正しい部分の解剖細胞、​​4）必要なマニュアルを開発解剖や文化への転送だけでなく時の損傷を防止するための器用さ。 特定の割球を操作するには、カーディナルの軸を識別できることが不?...

アフリカツメガエルの胚は、その卵が顕微鏡のアプローチを可能にするのに十分な大きさであるため、胚細胞がその特定の運命を獲得するメカニズムを識別するために広く利用されてきた。各セルには固有エネルギーストアを提供卵黄血小板の豊富な細胞内の供給が含まれているため、さらに、彼らは、培地の栄養補給を必要とすることなく、外部から開発しています。 1、2、3、4、5、6 -細胞の運命が決定されるメカニズムを研究するための重要な資産は、（32セルステージ2〜から）卵割期の割球の運命マップの包括的なセットです。これらのマップは、組織が標識された子孫が移入され、開発の後半にある単一の識別可能な割球および監視に検出可能な分子をマイクロインジェクションすることにより構築した。胚の枢機卿軸は確実に多くのエンブリーで同定することができるため、一貫性のある運命のマップが可能ですOS。植物半球ではないのに対し、第一に、すべての野生型胚において動物半球は、色素沈着です。第二に、受精時の精子のエントリは、将来の腹側に向かって動物半球の色素沈着の収縮を引き起こし、多くの胚では色素沈着の差が故に背側と腹側の側面を区別するために使用することができます。第三に、第一分裂溝は、ほとんどの胚における半ば矢状面を近似し、その結果胚の左右を識別するために使用することができます。運命マップも胚の大規模な人口全体の各割球が識別できるように規則的なパターンで自然に受精卵が頻繁に開裂するという事実に依存しています。内および色素沈着および切断パターンに関する卵のクラッチの間にばらつきがあるが、本明細書に記載の選択手順を使用すると、所定の運命を有する細胞が約90％の精度で識別することができます。 細胞の運命が決定することができますいくつかのメカニズムで胚発生時の採掘。そのような差別的継承細胞質mRNAまたはタンパク質などの本質的な要因は、早期のパターニングのいくつかの側面に貢献しています。たとえば、特定の母性mRNAは細胞は、生殖系列に寄与する内胚葉になったり、背側体軸（7日）に寄与するかを決定します。胚の情報伝達の中心から隣接セル以上の遠縁でローカルに提供外因性の要因は、ほぼすべての臓器系の特定の組織の種類とパターン形成を誘導するための責任を負うものとします。シグナリングセンターの例には、神経外胚葉と中胚葉のパターンを誘発原腸胚におけるオーガナイザー/ノード、および肢芽のパターンは前後軸という活動を偏光の帯があります。運命マップが異なる器官の前駆体を識別し、正常な胚でその子孫によって取ら発達経路を明らかにするが、それらは本質的な区別はできませんそして、それらの細胞に外因性の影響。彼らはまた、子孫胚の複雑な信号環境において分化細胞の完全な発達の可能性を明らかにしない。で培養した胚に由来する細胞の除去または2）;小説胚の場所に）1細胞の移植：2つの実験的なアプローチは、細胞の運命は、内因性の要因によって決定されたか、後に外部の要因によって影響されているかどうかをテストすることができます外因性シグナルの不在。 細胞が手動で分離するのに十分な大きさであるため、両方の実験的なアプローチは、 アフリカツメガエルで実現可能となっている。たとえば、多くの研究は、運命の変更（7日、8、9）をテストするために宿主胚における新規の場所に胚（細胞間相互作用を変更する場合）または移植された細胞からの単一細胞を削除しました。さらに、胚の異なる領域から細胞の数が少ないexplantingの第二のアプローチIアフリカツメガエル胚の細胞は、細胞内の栄養素ストア、卵黄血小板で埋められるため、外因性因子の非存在下で誘導組織の相互作用を解明するNTOの培養が可能です。したがって、彼らは、培地の栄養や成長因子の補充なしで定義された塩培地で数日間培養することができる。私たちは、その背の動物の球が母性遺伝mRNAが10、11に起因する神経と背側中胚葉組織を生成するために自律的能力を持っており、他は32細胞割球と中胚葉の仕様では、内因性及び外因性情報12の両方に依存して認められた表示するには、このアプローチを使用していた。外植体として、細胞を培養することの利点は、媒体は、外植セル12、13、14の運命に影響を及ぼす可能性のある細胞間の通信経路を決定するために定義されたシグナル伝達因子を補充することができることである。また、1割球はPRを注入することができます内因性mRNAの翻訳を防止するために過剰発現するmRNAの遺伝子を持つ、またはアンチセンスオリゴヌクレオチドを​​用いた文化にIOR。これらは、ゲインとロス·オブ·機能解析は、自律的に発現して運命のために必要とされる分子を識別することができます。外植片の運命を分析するには、特定の細胞種の同定は標準の遺伝子発現（ 例えば、in situハイブリダイゼーション 、RT-PCR）と免疫細胞化学的ア ​​ッセイにより行うことができる。このプロトコルは、胚細胞が特定の組織に発展する方法を制御内因性および外因性のメカニズムを区別するための簡単​​かつ強力な方法を提供します。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>アルミナ研磨フィルム</td><td>トーマス·サイエンティフィック</td><td> ＃6775E-38 </td><td>鉗子先端部の大規模修繕のためのコース（12μm）であり、 </td></tr><tr><td>アルミナ研磨フィルム</td><td>トーマス·サイエンティフィック</td><td> ＃6775E-46 </td><td>鉗子先端のシャープ罰金のための媒体（3μm）を、 </td></tr><tr><td>アルミナ研磨フィルム</td><td>トーマス·サイエンティフィック</td><td> ＃6775E-54 </td><td>鉗子先端の研磨のためのファイン（0.3μm）を、 </td></tr><tr><td>鉗子：デュモン、Dumoxel Biologie＃5 </td><td>ファイン科学ツール</td><td> ＃11252から30 </td><td>これらは、最初の購入時に鮮鋭化を必要としない微細なヒントがあります。耐腐食性、彼らがそうすることができるので、オートクレーブする。 </td></tr><tr><td>ゲンタマイシンソリューション</td><td>シグマ</td><td> G1397 </td><td>使用と同じ日に培地に入れる</td></tr></tbody></table>

blastomere explants to test for cell fate commitment during embryonic development

胚の細胞のすべてをトレース系統から構築運命マップは、組織は、胚の各セルから派生しているが明らかになった。運命マップは臓器の前駆体を識別するための、子孫細胞は正常な胚に、その臓器を移植それによって発達経路を解明するために非常に有用であるが、それらは、前駆細胞の完全な発達の可能性を説明したりするメカニズムを特定することはありませんその運命が決定される。細胞運命のコミットメントをテストするためには、実験的操作後に発現したものと無傷の胚（運命マップ）に子孫の細胞の正常なレパートリーを比較します。細胞の運命は、周囲の細胞環境にかかわらず、（コミット）が固定され、またはそのネイバーが提供する外部要因によって、それに影響を与えていますか？ アフリカツメガエル胚の包括的な運命マップを使用して、我々が識別する方法について説明し、隔離し、文化の単一卵割期の前駆体、blasto呼ばミアズ。このアプローチは、1つは、これらの初期の細胞は、彼らがそれらの隣接細胞との相互作用を必要とし、無傷の胚内でそれらの通常の環境で取得運命にコミットしている、または他のタイプの信号にさらされた場合、代替の運命を表現するために影響を受ける可能性があるかどうかを評価することができます。

正確に細胞の発生能を評価するために、このアッセイの能力は運命マップ1、2、3、4、5、6に基づいて、適切な割球を解離に依存している。したがって、それは続いて、 図2に示すように、定期的な切断パターンに準拠し、 図1に示すように、2細胞期に正しい色素沈着パターンを持つ胚を選択することが重要です。一つは、彼らが必要な切断段階に達するよう?...

Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

個々の胚細胞の運命は継承分子および/または隣接するセルからの信号により影響を受ける可能性がある。卵割期アフリカツメガエル胚の運命マップを利用して、単一割球は、細胞間相互作用と継承された分子の寄与を評価するために分離して培養のために識別することができます。

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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A Hierarchical Stem-Cell System: Blood Cell Formation

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15.2K ビュー.  The George Washington University. 個々の胚細胞の運命は継承分子および/または隣接するセルからの信号により影響を受ける可能性がある。卵割期アフリカツメガエル胚の運命マップを利用して、単一割球は、細胞間相互作用と継承された分子の寄与を評価するために分離して培養のために識別することができます。

ビデオ: Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

生物学

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

胚形成の間に機能的な冗長性をテストするための逆遺伝学的アプローチ

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

テラトーマ形成により、ヒト胚性幹細胞の運命のマッピング

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

ゼブラフィッシュにおける単一の細胞運命のマッピング

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

胚におけるプログラム細胞死を検出するためのLysoTrackerの使用は、胚性幹細胞を分化

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...