我々は、ブロード幹細胞研究センターのFACSを認識し、特に細胞選別を持つ彼らの助けのためにジェシカ·スコールズとフェリシアCodreaに感謝したいと思います。仕事がCIRM RN2-00904-1、K08 HL074229、米国胸部学会/ COPD財団ATS-06から065、懸念財団、UCLAのジョンソン総合がんセンター胸部腫瘍学プログラム/肺がんの胞子、カリフォルニア大学がんによって資金を供給された研究調整委員会とグウィンハーゼンチェリーメモリアル研究所（BG）。

手順の概要 1。気管切開 2。気管を洗浄し、それを切断 3。単一細胞懸濁液に酵素消化し、処理 4。 FACS用染色およびソート 5。 in vivoおよびin vitroモデルのために選別された細胞を処理する 1。気管切開<ol><li>ペントバルビタールの0.1 mg/0.2 ccの腹腔内注射でマウスを安楽死させる。 </li><li>腹壁を開いてカットし、腹部大動脈を露出し、それを解剖する側に腸を移動します。 </li><li>心臓や肺を露出するために両側の横隔膜と前胸壁を切断することによって胸を開きます。気管を露出するために脂肪と唾液腺を除去首にカットアップを続けます。 </li><li>胸腺を外し、下と後ろにその接続オフ肺を切った。 SEPARそれらの間に鉗子を挿入することで、食道から気管を食べその後、喉頭との接続上、その上部の大部分で気管を切った。その後、鉗子で気管を保持し、その添付ファイルからそれを解剖し、心臓と肺の専用ブロックでそれを除去するために下向き継続。左右の主気管支のほとんどが含まれるように門の近くに心臓や肺を切り取った。 </li><li>氷の上に培地を収集含む小さなシャーレに気管を置きます。 </li></ol> 2。気管を洗浄し、それをカッティング<ol><li>解剖顕微鏡の下で、それに接続された他のすべての組織から気管をきれいにする非常に細かい点鉗子（第5号）及びVannasテュービンゲンはさみ（FST 15003から08 finescience.com）を使用します。これは含まれています：リンパ節、反回神経、脂肪、甲状腺、残りの肺血管を。気管の上部には、SMGの最大の束を損なうことなく、大きな喉頭軟骨を除去するために、特別な注意を必要とそれは輪状軟骨と第一気管軟骨輪（C1）は4の間に提出されています。 </li><li>上部とC4とC5の間下部に気管を切断してから、気管の管腔を介して両方の部分を切り開いた。 </li></ol> 3。単一細胞懸濁液に酵素消化および処理（SCS） <ol><li> 5前述のように、30分間室温（RT）でエッペンドルフチューブ内16 Uディスパーゼ1ml中に気管の下部をインキュベートする。その後、室温でさらに20分間DMEM中で0.5 mg / mlのDNase Iを追加します。 </li><li>滅菌シャーレに新鮮収集培地に消化気管を外し、解剖顕微鏡で表面上皮を取り除くことにしました。 50mlコニカルチューブに取り除か上皮を含む培地を転送するためにピペットを使用しています。 </li><li> 4℃、1,000×gで取り除か上皮をスピンダウン上清を除去し、目を揺らしながら0.1％トリプシン/ EDTAとインキュベートを追加37の電子管℃で30分間インキュベーター（200 rpm）を振ること。インキュベーション後、1,000μlの先端を使用して、塊を分割し、単一の細胞懸濁液を形成するためにピペッティングします。 </li><li> 0.15％プロナーゼの1ミリリットルを含む円錐管の上部気管を置きます。その後、4時間、4℃でインキュベートする。 </li><li>チューブボルテックスした後、新鮮な培地に集気管を置き、表面上皮（SE）の完全な剥離があることを解剖顕微鏡下で確認してください。 </li><li> SMGコンパートメントを開くために細かいハサミで気管をミンチした後、低速振盪しながら室温でプロナーゼで1時間以上インキュベートします。 </li><li>気管組織をスピンダウンし、プロナーゼを削除するには、上記のように振とうしながら30分間トリプシン/ EDTAとインキュベートし、37℃で0.1％を加算します。 </li><li> 10mlのシリンジを使用して20G、23Gと26Gの針を通過消化細胞塊と腺の残りのチャンク。前に、それぞれの針のサイズで継代を数回繰り返し辞任。今では、ほとんどの細胞は、SCSにする必要があります。 </li><li>その後、ストレーナを洗浄し、細胞をスピンダウンし、40μMストレーナーを通して懸濁液をろ過する。それから媒体の適切な音量で再構成する細胞は、細胞をカウントし、染色し、FACSソーティングにすぐに進みます。私たちは、これが生存率に影響を及ぼし、深刻な効率性を形成する球を減少させることがわかったので、4℃で、または数時間氷上で細胞を放置しないでください。 </li></ol> 4。 FACS用染色およびソート<ol><li>その後、室温で15分間、一次抗体、ヤギおよびラットTrop2 ITGA6を持つ細胞の残りの部分を染色する染色されていないし、単一ステンド補償チューブを準備するために十分な細胞を取る。その後、PBSで洗ったり、培地を回収し、上清を除去し、非常に慎重に、細胞ペレットを破る。 </li><li>室温で10分間、暗所でインキュベート適切な二次抗体を用いて染色、洗浄、上清を除去した後、ペレットを破る。 </li><li> FACSにあなたの電圧と補償、破片出門、非常に小さな細胞、細胞ダブレットと死細胞を調整した後、ARIA、。 SMG細胞の20〜50％がTROP2 +6になりますしながら、SEからのほとんどの細胞はTROP2正でなければならない。 </li><li>あなたのソートTrop2用ゲート+ ITGA6 SMG（管細胞）からSEから+細胞（基底細胞）とTrop2 +の細胞（+ /-ITGA6陽性細胞）を設定</li></ol> 5。ために選別された細胞の処理： 体外培養および in vivo モデルにおける 5月9日の文化を形成してin vitroでの球で 5.1 <ol><li> 5万選別された細胞までを含む完全培地10,11100μlと&quot;マトリゲル減少成長因子&quot;の100μlを混ぜます（ 表1を参照）は、次にトランス24ウェルの上室にこの混合物を入れた。その後、37℃、下部チャンバーに完全培地400μlを入れ、インキュベート℃に隔日下部チャンバー内の媒体を変更し、マトリゲルの余分100μlを加える毎週またはそれは薄くなった場合、以前の。 </li></ol> in vivoモデル 5.2 <ol><li> 50μlあたり5,000-10,000細胞の濃度でマトリゲルと1:1で混合完全培地中で再構成するソート細胞ペレットを。 </li><li>受信者C57/BL6マウスを麻酔。鎮痛剤としてsubcutaenouslyカルプロフェン（5 mg / kg）を投与する。背中の上部にある中央の長手方向の皮膚切開を行い、次にその背中を殺菌し、剃る。切開は脂肪パッド注射部位だけでなく、注射後ブレブ形成を識別するのに十分な大きさでなければなりません。アクセスと可視化を最大化するために両側胸壁の皮膚と背中の間に袋を広げてはさみの鈍側を使用します。 </li><li>肩甲骨が動いて確認するまで、後方マウスの前肢を押すことにより、肩甲骨の内側縁を識別します。 300μlのBDのインスリン注射器に細胞懸濁液50μlを取り、ちょうど肩甲骨へ内側へと横方向の脂肪パッドにそれを注入する椎骨。あなたはあまりにも深いそうでなければブレブを見ることができるはずです。クリップやステッチ皮膚切開後、反対側に同じことを繰り返します。マウスを確認し、48時間1日1回（5 mg / kgを、サウスカロライナ州）カルプロフェンを与える。 </li><li> 3週間後、pentobarbatol（0.1 mg/0.2 CC、IP）にマウスを安楽死させる、上記のように皮膚を開き、周りに形成され、拡張された可能性のある組織構造の欠落を避けるために安全マージンの両側から全体の脂肪パッドを解剖。パラフィン12にPFAとembedで固定してください。細心の注意は、トリミングや構造をバイパスしたり、見落とさないブロックを切削中に採用すべきである。ベストプラクティスは、一旦、4μmのスライスをカットスライドガラスの上に置き、ブロックかどうかのこの部分の任意の &quot;上皮構造&quot;があるかどうか確認するために、顕微鏡下でそれを調べる40μmをトリミングすることである。見たものが何もない場合は、もう一度トリミングし、この手順を繰り返します。 </li></ol> 6。代表的な結果 STRI図1Aに示すように、光学顕微鏡で見える裸に気管になり、マウスの気管pping。 図1Bは、上皮がブドウの組織と似た房から解放された粘膜下腺ストリッピング後の気管の明視野ビューを示しています。 プロナーゼはITGA6エピトープを削除し、TROP2ダクト細胞がITGA6に分離できない理由はここにある+と - 集団。しかし、ITGA6エピトープを保持ディスパーゼは、非常に短いインキュベーション時間であってもすべての表面上皮および管細胞を消化するため、3基底細胞からダクトを分離するために使用することはできません。単一の細胞懸濁液のフローサイトメトリーは、 図2Aに示されている前方散乱と側の散布図が表示されるはずです。残りの部分から管細胞の良好な分離気管細胞は、蛍光活性化細胞sortiためのフローサイトメトリーでTrop2抗体で見られるかどうか図2Bに示すように、NG（FACS）。球は、文化の中で、約1週間後にマトリゲルに表示され、 図3に示すように、外観に密集している必要があります。このプロセスの効率は1-2％であり、単一セルの順序がまだマトリゲル中に存在する可能性が高いと自己再生能力を持っていない粘膜下腺管から細胞を表すということです。 図4に示されている粘膜下腺様構造の形成におけるマウスの脂肪パッドの結果にダクト幹細胞の注入。これらはあっても、H＆E染色せずにブロックで見られている。これらは球状であり、中央の内腔を有する。多くのクロスセクションでは、これらの上皮構造を逃さないようにするために、脂肪パッドを介して行う必要があります。 <img alt="図1" src="/files/ftp_upload/3731/3731fig1.jpg" /> 図1：気管上皮の酵素消化。表面上皮細胞の除去はBLACと共に示されているk個の矢印、粘膜下腺管の細胞の除去は、緑色の矢印で示されています。光学顕微鏡で見える裸にされた気管はディスパーゼ消化の30分後に、プロナーゼ消化の4時間後に表示されます。 <img alt="図2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> 図2A。私。上皮表面が剥離した後、マウスの気管から代表的な前方散乱および側方散乱プロットを示す単一の細胞懸濁液のフローサイトメトリー。 II。マウスの気管から管細胞におけるTROP2表現するための代表的なFACSプロット。 <img alt="図2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> 図2B。私。代表的な前方散乱および側方散乱プロットを示したマウス気管の下部3分の2の剥奪表層上皮からの単一細胞懸濁液のフローサイトメトリー。 II。 suコマンドでITGA6式の典型的なFACSプロット上皮をrface。 ⅲ。緑の中の基底細胞集団のゲーティングを示す背面表面上皮から前方および側方散乱のもう一つの代表的なFACSプロット。 IV。典型的なITGA6とTROP2が緑色に細胞集団を表現する。 <img alt="図4" src="/files/ftp_upload/3731/3731fig3.jpg" /> 図3の球の開発のタイムコース。 A.ダクト球。球は培養液中で約1週間後にマトリゲルに表示する必要があると、ほとんどすべての外観が密集している。球体を形成していない単一細胞も見られている。 （スケールバー= 50μm）である。 B.基礎球は外観が大きく、管腔である。球体を形成していない単一細胞も見られている。 （スケールバー= 50μm）である。 <img alt="図4" src="/files/ftp_upload/3731/3731fig4.jpg" /> 図4：粘膜下腺様構造の形成におけるマウスの脂肪パッドの結果にダクト幹細胞の注入。代表的な構造脂肪パッドに示されており、一部はアルシアンブルー過ヨウ素酸シッフ染色とムチン（ライトブルー）の製造示されています。

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特別な利害関係は宣言されません。

気道からダクトおよび基底細胞を単離するために、この手法は、気道の修復及び再生及び気道疾患の我々の理解を向上させるために重要である。ここで説明する手法は、いくつかの重要なステップがあります。第一は、最適化された酵素消化期間です。第二は、細胞の毛刈りを防止するだけでなく、細胞塊を分割するために徐々に高くゲージ針で継シリアルを介して単一細胞懸濁液を作成す?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>媒体10を完了して下さい ： DMEM-F-12、50/50、1X） </td><td>メディアテック</td><td> 15から090-CV </td></tr><tr><td> HEPES（15mMの） </td><td>インビトロジェン</td><td> 15630 </td></tr><tr><td>炭酸水素ナトリウム（3.6ミリメートルまたは0.03％） </td><td>インビトロジェン</td><td> 25080 </td></tr><tr><td> L-グルタミン（4mMの） </td><td>メディアテック</td><td> 25から005-Clを</td></tr><tr><td>ペニシリン（100 U / ml）を</td><td>メディアテック</td><td> 30から001-CI </td></tr><tr><td>ストレプトマイシン（100μgの/ M） </td><td>メディアテック</td><td> 30から001-CI </td></tr><tr><td> アムホテリシンB（0.25μg/ ml）を</td><td> ロンザ </td><td> 17-836R </td></tr><tr><td>でsulin（10μg/ ml）を</td><td>シグマ</td><td> I6634 </td></tr><tr><td>トランスフェリン（5μg/ ml）を</td><td>シグマ</td><td> T1147 </td></tr><tr><td>コレラ毒素（0.1μg/ ml）を</td><td>シグマ</td><td> C8052 </td></tr><tr><td>上皮成長因子（25 ng / ml）を</td><td> BD </td><td> 354001 </td></tr><tr><td>ウシ下垂体抽出物（30μg/ ml）を</td><td>インビトロジェン</td><td> 13028-014 </td></tr><tr><td>ウシ胎児血清（5％） </td><td>フィッシャー</td><td> SH3008803HI </td></tr><tr><td>レチノイン酸（0.05μM） </td><td>シグマ</td><td> R2625 </td></tr><tr><td>マトリゲル減少した成長因子</td><td> BD </td><td> 354230 </td></tr></tbody></table> 表1は、。メディアコンポーネントを完了します。 <table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </TD&gt; <td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>プロナーゼ</td><td>ロッシュ</td><td> 10165921001 </td><td> 0.15パーセントで使用： 4 AT-O / N°C消化合計気管細胞を（ALI培養用）を単離する -4時間消化4℃隔離するためにSMG </td></tr><tr><td>ディスパーゼ</td><td> BD Biosciences社</td><td> 354235 </td><td>室温で30分：16号機で使用</td></tr><tr><td> DNase Iを</td><td>シグマ</td><td> DN25 </td><td> 0.5 mg / mlで使用される： 室温で20〜30分間</td></tr></tbody></table> 表2。気管の酵素消化のために使用される酵素。

isolation of basal cells and submucosal gland duct cells from mouse trachea

大気道が1と我々は息毒素や感染性病原体による損傷することが影響を受けやすい環境と直接接触しており、。大気道が故に、私たちの体を守るための効率的な修復機構を必要とします。この修​​復プロセスは、気道内の幹細胞から発生し、気道から、これらの幹細胞を単離する修復および再生のメカニズムを理解する上で重要である。これは、気道疾患2につながることができます異常な修理を理解するためにも重要である。この方法の目的は、マウス気管粘膜下腺管から新規幹細胞集団を単離するために、粘膜下腺の3修復および再生のメカニズムを識別するために、in vitroおよび in vivoモデル系において 、これらの細胞を配置することです。この生産は、大規模な気道3からダクトおよび基底幹細胞を分離し、アッセイするために使用できる方法を示していますこれは、私たちを許可します嚢胞性線維症、喘息および慢性閉塞性肺疾患などの気道の疾患を研究する。現在、粘膜下腺管細胞を単離するためのメソッドはありませんし、粘膜下腺の再生を研究するin vivoモデルでは存在しています。

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

ここでは、マウスの気管から基礎および粘膜下腺管細胞を単離するための我々のプロトコルを示しています。また、作成するために背マウス脂肪パッドに幹細胞を注入する方法を実証<em生体内で&gt;</em粘膜下腺の再生&gt;モデル。

マウス気管から基底細胞および粘膜下腺管細胞の単離

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

isolation-basal-cells-submucosal-gland-duct-cells-from-mouse

Research

JoVE Journal

Biology

13.4K ビュー.  David Geffen School of Medicine at UCLA. ここでは、マウスの気管から基礎および粘膜下腺管細胞を単離するための我々のプロトコルを示しています。また、作成するために背マウス脂肪パッドに幹細胞を注入する方法を実証モデル。

ビデオ: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4 +ミルテニーMACS精製を使用してマウスのリンパ節からT細胞の分離

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

マウスの眼から網膜幹細胞の単離

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods Article

マウス気管から基底細胞および粘膜下腺管細胞の単離