Name: derivation of t cells in vitro from mouse embryonic stem cells
Uploaded: 2014-10-14T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

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The authors have nothing to disclose.

OP9-DL1共培養系幹細胞からの血液細胞型の発達中のさまざまな遺伝子産物の役割を研究するために利用されている。8,12,13また、遺伝子調節DNAの機能を研究するための有効なモデルを証明して細胞分化の間に。9,14全体のマウスモデルの代替としてこの方法を使用すると、造血における多くの基本的な質問に対応した実験の時間とコストでかなりの節約をもたらすことができる。しかし、これらの目的のためにこのプロトコルの適合は、これらの手順で使用されるであろうMESCクローン間の潜在的な変動性の認識が必要である。公開されたプロトコルのタイムラインは、手入れの行き届いた、非操作さMESCライン、平均値から期待できる。7また、キメラマウスの作製のために選択されMESCクローンは通常、より高品質のものであり、OP9共培養では非常に堅牢に実行されます。一方、MESCクローンが安定から直接出現異所的に組み込まれた導入遺伝子を用いたトランスフェクションは、平均から平均以下の範囲の初期分化効率のさまざまな程度を表示します。ここに記載されているプロトコルは、in vitroでの造血の誘導に先立ってこれらの潜在的な違いを均等に5日目の通過段階の戦略的な1-2日の遅延を提供します。 8日目の通路は、このプロトコルの実行がエラーの最も可能性を秘めていれるステップである。忍耐、練習や注意深い観察がOP9単層の中断を最小限に抑えながら、HPCの収穫を最適化する技術を開発することを可能にします。キー5日目および8日目の手順を超えて、重要なパラメータは、プロトコルにおいて使用される試薬に細心の細胞培養の維持、（上述のように、特にOP9細胞）および特別な注意を必要とする。最も重要な試薬はFBSである。 FBSの明確な多くは、この手順をサポートする能力が著しく変化します。複数のロットは、Oで試験されなければならないこのアッセイにおいて強固な差別化を生み出すかを決定するRDER。 この共培養系には限界がある。たとえば、このモデルは、単一陽性CD4細胞の分化をサポートしていません。この理由の一つは、OP9細胞における主要組織適合遺伝子複合体（MHC）クラスII抗原提示インフラストラクチャの発現の欠如である。MHCクラスIIによる抗原の1の細胞表面提示は、CD4 SP細胞の適切な発達に必要とされる。成熟および未熟CD8のSP胸腺細胞ステージの組み合わせを表して出てくるんCD8 SP細胞。MESCの1また、移植の成功は、マウスにT細胞の前駆細胞に由来する胎児胸腺器官培養を通して前に通過する必要があります。1それにもかかわらず、この技術は、その証明されているVで探索する以前は可能であった血液や免疫系の発達における細胞および分子の質問の両方への強力な調査手法として期待IVO。このように、脇にこれらの質問に対するより迅速な進歩を遂げるための新規な方法を提供するから、OP9-ESC共培養系の広い採用は、実験動物の使用を削減するより広範な影響を与えることになる。

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table><tbody><tr><td>DMEM</td><td>Corning</td><td>15-013-CV</td><td></td></tr><tr><td>Stem cell qualified FBS</td><td>Gemini</td><td>100-125</td><td>Heat inactivated</td></tr><tr><td>Penicillin/Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-alanyl L-glutamine</td><td>Corning</td><td>25-015-CI</td><td></td></tr><tr><td>HEPES buffer&amp;nbsp;</td><td>Millipore</td><td>TMS-003-C</td><td></td></tr><tr><td>Non-Essential Amino Acids</td><td>ThermoScientific</td><td>SH30853.01</td><td></td></tr><tr><td>Gentamicin Regent Solution (50 mg/ml)</td><td>Life Technologies&amp;nbsp;</td><td>15750-060</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol (55 mM) in DPBS</td><td>Life Technologies&amp;nbsp;</td><td>21985-023</td><td></td></tr><tr><td>Filter Unit</td><td>Millipore</td><td>SCGPU05RE</td><td>0.22 &amp;mu;m PES membrane</td></tr><tr><td>Cell Culture Grade Water</td><td>Corning</td><td>25-055-CM</td><td></td></tr><tr><td>&amp;alpha;-MEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>12000-022</td><td>Powder, reconstitute per manufacture recommendation</td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>FBS</td><td>ThermoScientific</td><td>SH 30396.03</td><td>Testing of individual lots required&amp;nbsp;</td></tr><tr><td>Dimethyl Sulphoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Recombinant Human Flt-3 Ligand</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>PeproTech</td><td>217-17</td><td></td></tr><tr><td>LIF&amp;nbsp;</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Utrapure water with 0.1% gelatin</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>MEFs mitomycin C treated</td><td>Millipore</td><td>PMEF-CF</td><td>Any mitotically arresteded MEFs can be used</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Cell strainer (40 &amp;mu;m)</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Tissue culture dish 100 x 20 mm</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Multiwell 6-well</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td><td>BD Falcon</td><td>352235</td><td>Tubes for FACS&amp;nbsp;</td></tr><tr><td>ES R1 cells</td><td>ATCC</td><td>SCRC-1011</td><td></td></tr><tr><td>OP9 cells</td><td></td><td></td><td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td></tr><tr><td>OP9-DL1 cells</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td>Cells can be requested from the Z&amp;uacute;&amp;ntilde;iga-Pfl&amp;uuml;cker laboratory.</td></tr><tr><td>FlowJo software&amp;nbsp;</td><td>Tree Star</td><td></td><td>FACS data analyses</td></tr><tr><td>Flow Cytometer</td><td>BD</td><td></td><td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td></tr></tbody></table>

derivation of t cells in vitro from mouse embryonic stem cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

LIFの存在下で、MEF上で増殖させた場合、たmESCは、未分化状態に維持することができる。理想的な条件下では、それらは位相差顕微鏡（ 図1）に光沢のあるハローに囲まれた細胞のコンパクトなコロニーとして見える。これらの培養物を毎日監視する必要があります。細胞のコンフルエンスに応じて、メディアを変更することができ、または細胞が分割することができる。隣接MESCコロニーは互いに接触点に来るべきではありません。未分化たmESCの正常な、健康な培養物は、 インビトロ T細胞分化に必須の出発点である。共培養を開始すると、それは細胞が過密することなく、完全にコンフルエントにすることをお勧めします。 OP9単層上に細胞を播種するために回収した細胞の大部分が（むしろフィーダー細胞以外）MESCであるようにするためである。合流点での主な違いは、0日に区別される各種MESCクローン間で観察されている場合、それはTISに予め付着によりMEFにを除去することがベストです共培養シードにたmESCをカウントする前に、（完全なES細胞培地を使用してステップ3.4.3-3.4.6のように）培養皿を訴える。 （セクション2.4を参照）OP9細胞は、先行共培養開始を維持しなければならない。加えて、OP9細胞は長期培養および/ ​​またはそれらの増殖率の顕著な増加にそのプロパティの一部を失うと考えられている7が 1を超えて分割比率の回避：OP9細胞の維持の間、5、および6週間後に連続OP9培養を破棄、これらの落とし穴を避けるのに役立ちます。 初期細胞播種およびセル転送共培養する段階（0日目、5、8、12、16）で、OP9単層を合流点の最適な範囲内にあるべきである。 図2（図2A）、低および高（ 図を示している図2（b））共培養単層として使用するための賢明OP9細胞コンフルエンスの範囲を終了します。過剰コンフルエントなプレートの例を図5に示すURE 2Cを。最適なコンフルエンスに維持しないと、大量に、負に共培養に影響を与えることができる、脂肪細胞（ 図2D）の形成を誘導することができる。 中胚葉形成をOP9細胞に好まれているので、0日目に、共培養はOP9細胞ではなく、OP9-DL1細胞上で開始されます。細胞は、T細胞産生を誘導することが8日目に開始OP9-DL1細胞単層上にプレーティングする。いくつかのMESCクローンが視覚的に共培養の5日目までに中胚葉様コロニーの堅牢かつ定量的（〜90％）の形成（ 図3A-B）を表示しますが、他の人は、このステップ（ 図3C-F）で遅延を表示することができます。顕微鏡下で、このアッセイにおいて中胚葉由来の様コロニーを可変ワゴンホイールまたはクレーター（ 図4A）またはスターバーストまたは小花（ 図4B）に似ているとして記載されている。 公開されたOP9-DL1プロトコルは、定量的な中胚葉フォーマのノルムを反映しているが共培養の5日目によるションは、7私たちは 、すべてのESCクローンがこの時間枠内でそのノルムを達成しないことがわかります。これらのケースでは、5日目に培地を変更し、「5日目」は、細胞の収穫/転送プロトコルを実施する前にさらに1〜2日待ってください。この遅延の目的は、転送する前に視覚的になる中胚葉へ〜ES細胞コロニーの80〜90％を可能にすることである。これは80〜90％の図は、共培養皿でコロニー形態の単純な位相差顕微鏡検査によって厳密に視覚的な基準を反映して識別できることを明確にすることが重要です。それは、図4に示したものに似ているESCコロニーの割合を指す。それはいくつかのESCクローンであっても二日間の遅延の後に、この視覚的判定基準に到達しない可能性がある。以前に記載されているような場合には、あるFlk-1 +細胞について細胞選別、フローサイトメトリーを用いて血液形成中胚葉前駆体を富化することが可能である。いずれにせよ6,11、saのためにKE命名の利便性、私たちは &quot;時計をリセット」と「5日」と中胚葉様コロニー移転の日を参照してください。 複数のESCクローンが並列に分化されているとき、それは他の人が遅れている間、いくつかの共培養は、時間に5日目通過のための準備ができていることが可能である。この場合には、より低速のクローンが他の人に追いつくまで、すべての共培養の通過を遅らせることをお勧めします。遅延は、「オンタイム」の共培養に害をしないようだが、遅いクローン大量のその後の分化に利益をもたらす。 8日目（ すなわち 、中胚葉コロニー移転後​​3日）は、HPCのの出現および蓄積を見ている。 HPCは緩くOP9フィーダー細胞（ 図5）に付着している細胞の光沢のあるクラスターとして表示されます。残しながら慎重に洗浄操作を、ピペットや皿上のメディアを使用して、切り離し、HPCのを収集するOP9単層、ほとんど無傷で（ 図6A-C）。これはおそらく、プロトコルの中で最も技術に敏感なステップです。これは、フィーダー層の中断を最小限に抑えるとのHPCの最大収穫の所望のバランスを達成するために、適切な運動とメディア吐出圧を見つけるためにいくつかの練習が必要です。 OP9単層の一部がリフトオフした場合の浮遊単層の大半はフィルタリング後40μmのナイロンメッシュで捕捉されますので、それは。 図6Dが必要以上に大きい単層の破壊につながる過剰なピペッティングした後、単層を示し、許容可能である。この洗浄工程の間に、それはすべてのHPCが収集されたか否かを顕微鏡下でチェックし、それに応じて、洗浄圧力を調整することが重要である。 共培養の進行は、フローサイトメトリーによってモニターすることができる。特にESCの非赤血球造血子孫を分析するためには、生きたCD45 +細胞上の第一ゲートすることが重要です。これは、ステップの画面外分析からのOP9細胞を、DAPIまたは他の核染色色素の使用は、非生存細胞の排除を可能にしている。 12日目では、小さくて丸い、光沢のある細胞の多くのクラスターが表示されるはずです。これは、この段階で細胞を計数する必要はない。しかし、私たちは生きている、ゲート制御フローサイトメトリーのイベント数はよく、12日目の共培養あたり1〜3×10 5の範囲が一般的であることを観察した。フローサイトメトリーは、赤血球が得られるOP9単層上で差別化生依存性細胞（CD45のNEG、TER119 +）および単球（CD45 +、CD11bのハイ ）の系統（ 図7A）の分析。 OP9-DL1の単層上での差別のライブ、CD45 +細胞の分析は、CD4 / CD8ダブルネガティブ（DN）-1（CD44 +、CD25のNEG、CD4のNEG、CD8の負 ）に特徴的なマーカーを明らかにし、DN2（CD44 +、CD25 +、必要がありますCD4のNEG、CD8の負 ）ステージT細胞（ 図7B </stro<html> NG&gt;）。 16日目では、培養中の小さくて丸い、光沢のある細胞の量の有意な増加があります。 OP9-DL1sでは、T細胞分化製品はDN3（CD44のNEG、CD25 +、CD4のNEG、CD8の負 ）とDN4（CD44のNEG、CD25のNEG、CD4のNEG、CD8の負 ）の段階へと進行する。いくつかのDP（CD4 +、CD8 +）T細胞はまた、16日目（ 図7B）の出現を開始することができる。 HPCははOP9細胞上に播種OP9-DL1-MESC共培養の第20日では一日16でB細胞（CD19 +）が得られ、DP T細胞と現在シングルポジティブ（SP）CD8 + T細胞（大容量のがあるでしょう図7B）。図8は、共培養中に、上記の手順の重要なステップを要約図を提供し、提案され、分析の時点。 ghres.jpg &quot;/&gt; MEF単層（100×倍率）の上に成長する未分化たmESCの図1の位相差顕微鏡像（シャープエッジコロニー）。 <img alt="図2" fo:content-width="3in" src="/files/ftp_upload/52119/52119fig2highres.jpg" width="600" /> 図2：オーバーコンフルエントOP9単層のOP9単層の密集度の位相差顕微鏡像（A）共培養通路/播種（40倍の倍率）のための賢明最低と（B）最高密集度レベル（C）の例（40X倍率）。（D）オーバーコンフルエントOP9単層（200X倍率）脂肪細胞形成（大、小胞を含有する細胞）である。 <img alt="図3" src="/files/ftp_upload/52119/52119fig3highres.jpg" /> 図3：位相コントラストmicroscop共培養の&quot;5日目&quot;での中胚葉様コロニー形成のyの画像。（A）、40Xおよび（B）&gt; 90％の中胚葉分化コロニーとの共培養プレートの100X景色。これらのプレートは、5日目を通過させるための準備ができています。転送（C＆E、40X倍率、D＆F、100倍の倍率）の前に1-2日の延期を必要とする5日目の共培養プレートの（CF）の例。 <img alt="図4" src="/files/ftp_upload/52119/52119fig4highres.jpg" /> 図4：共培養の5日目に中胚葉様コロニーの形成の多様な位相差顕微鏡像（A）「クレーター」またはと呼ばれるコロニー形態（B）と呼ばれるコロニー形態」ワゴンホイール。 &quot; 「スターバースト」または「小花」（200×倍率）である。 OP9単層（40X倍率）に小さな、丸い、光沢のHPCのクラスタと8日目の共培養プレートの図5の位相差顕微鏡像。 <img alt="図6" src="/files/ftp_upload/52119/52119fig6highres.jpg" /> 図6：共培養8日目のHPCコレクション後OP9単層（AC）のHPCを収穫する入念な洗浄の後OP9単層の破壊の適正範囲（D）日までにかけて破壊されているOP9単層の一例。 8 HPC収穫手順。 <img alt="図7" src="/files/ftp_upload/52119/52119fig7highres.jpg" /> 図7：代表的なフローサイトメトリー（FACS）の中に特定の時点で解析in vitroでの MESC分化。赤血球のための（A）染色（左）、単球（中央）、およびB細胞示されるように、12日目または16にMESC-OP9共培養（右）製品。（B）の示された時点でのT細胞を開発するための染色MESC-OP9-DL1共培養。免疫表現の詳細な説明のためのテキストを参照してください。 <img alt="図8" src="/files/ftp_upload/52119/52119fig8highres.jpg" /> MESC-OP9共培養手順のステップの図8図（A）共培養の最初の8日間の主要な細胞転写工程。細胞のおよその数は日をゼロにOP9細胞上に播種し、5が示されている。（B）8日目の転写工程と共培養の重要な時点で、フローサイトメトリーによって検出された期待細胞分化の製品を。免疫表現の詳細な説明のためのテキストを参照してください。

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

10.9K ビュー.  City University of New York. Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

ビデオ: Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

生物学

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺の特性を用いてマウス胚における前駆細胞をセトリング<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;アッセイ

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

発生生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

マウス誘導多能性幹細胞由来腫瘍抗原特異的胸腺移民を生成する三次元胸腺培養システム

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

がん研究

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

マウス着床前胚幹細胞の誘導

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

胚盤胞からマウス栄養膜幹細胞の導出

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

<meta charset="utf8"></head><body>フローサイトメトリーを用いたナイーブCD4+ T細胞からのTh17細胞分化

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>著者スポットライト:サイトカイン濃度調節を用いたTh17細胞の高純度In vitro分化の達成

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

マウスにおけるナイーブCD4+ T細胞の病原性Th17細胞へのin vitro分化

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

T細胞の導出

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