私たちは、彼女の貴重な援助のためにローラクラークに感謝。幹細胞ネットワーク、CIHRとNIH：この作品は、NCEによってサポートされています。

1。解剖ソリューションと酵素液を準備する<ol><li>事前に人工脳脊髄液（ACSF）と無血清培地を作成し、冷蔵。 </li><li>キヌレン酸（0.2 mg / mL）を秤量し、それが容易に溶解しないので、事前に37℃の水浴中でヒロACSFを10 mLに溶かす。 </li><li>トリプシン（1.33 mg / mLの）とヒアルロニダーゼ（0.67 mg / mLの）一、15 mLの試験管内の場所を秤量し、必要になるまで-20℃で保管してください。このチューブにキヌレン酸のソリューションを追加し、使用直前に22μmのシリンジフィルターを使用してフィルタ。 </li><li>トリプシンインヒビター（Ovamucoid：1 mg / mL）を秤量し、22μmのシリンジフィルターを使用して暖かい無血清培地とフィルターで溶解する。 </li><li> FGF2（10 ng / ml）およびヘパリン（2μg/ mL）を含む無血清培地：メッキのメディアを作成します。 </li></ol> 2。マウスの眼から網膜幹細胞を単離する<ol><li>網膜幹細胞の単離は、初代培養の実験に特化した特定の無菌室で行われます。この手順を開始する前に、滅菌解剖器具をレイアウトして解剖顕微鏡と冷光源を設定し、。それぞれのフードには、ステップ間の楽器を滅菌するためのホットビーズ滅菌器を持っています。 </li><li>我々は、承認された動物の倫理のプロトコルに従って屠殺したマウスからそれを除去した後、人工脳脊髄液（ACSF）を含む滅菌ペトリ皿にすぐに配置されている眼から網膜幹細胞を分離します。 </li><li>解剖顕微鏡下で、鉗子で目を洗浄：髪の毛と角膜/強膜の境界線に接続されている結合組織を取り除く。その後、ACSFを使用して新しい料理に目を移す。 </li><li>優しく鋸歯鉗子で固定目を押さえながら、眼筋を除去するために角度付きのマイクロ解剖ハサミを使う。それがまだ目に添付されている場合だけでなく、視神経を削除します。このプロセス中に目をつぶししないようにしてください。 ACSFを使用して新しい料理に目を移す。 </li><li>次の使用は、はさみが半分に目をカットするためのマイクロ解剖湾曲：カットが開始された視神経を振り返っ満たし、角膜の中央を通る視神経とカットから始まります。これは次のステップを容易にするため、眼の2つのサイズがほぼ同等である場合、それは理想的です。 </li><li>離れて2つのアイ半分剥離優しく、二つの鉗子を使用して角膜を開催。レンズ、内臓、および目のシェルから神経網膜を取り外して廃棄。 ACSFで新しい皿にシェルを転送する。 </li><li>今、私たちは、毛様体上皮を分離する準備が整いました。開始するには、東洋の目のシェルでは、その角膜があなたの右と網膜色素上皮（RPE）になっていることを左手にあります。ゆっくりとRPE側のストレートピンセットで目のシェルを突き止める。 </li><li>次に、静かにメスを押し下げはなく、製材の動きを使って、眼の毛様体上皮から角膜と虹彩をカットするためにメスを使用してください。 </li><li>その後、離れてRPEから毛様体上皮をカット。 ACSFを含む新しい35 - mmディッシュに毛様体上皮のストリップを転送するために、非鋸歯状の鉗子を使用してください。 </li><li>同じように、他の眼のシェルから毛様体上皮を分離する。 </li><li>両方の毛様体上皮細片を収集したら、ディスパーゼの2 mLを含む、35 mmディッシュにストリップを移す。 10分間、37℃インキュベーターでストリップで皿を置きます。 </li><li> 10分後、フィルタ処理されたトリプシン、ヒアルロニダーゼ、およびキヌレン酸2 mLを含む皿にディスパーゼからストリップを転送する。 37皿を置き° Cで1​​0分間。 </li><li>今解剖顕微鏡に戻ります。ストレートピンセットで強膜を押しながら、静かに離れて強膜から毛様体上皮の傷が付かないように湾曲した非鋸歯状の鉗子の底を使用してください。 </li><li>皿から強膜を取り除く。皿に残されるべきものは、現在の関心と酵素液の細胞である。 </li><li>ファイアーポリッシュ綿プラグのピペットを使用して、14 mlのチューブにこのソリューションを転送する。ピペットのinとoutソリューションを強制することにより上皮細胞を分解するために、このソリューションを30回ひいて粉にする。 </li><li> 1500rpmで5分間、チューブを遠心する。細胞はこの段階でチューブの底を抜けになりやすいので、遠心分離が終了すると、慎重に遠心機からチューブを取り外します。 </li><li>ゆっくりとファイアーポリッシュピペットを用いて上清の大部分を吸引し、細胞に無血清培地でトリプシンインヒビターの1 mLを加える。それは、単一細胞懸濁液になるまで、約50倍のサンプルをひいて粉にするために小さな孔綿栓したピペットを使用してください。 </li><li> 1500rpmで5分間再びチューブを遠心する。 </li><li>上清を除去し、メッキの培地1mLに交換してください。細胞を再懸濁するためにファイアーポリッシュガラスピペットを用いて軽く砕いて粉にする。 </li><li>カウントの後板その24 - ウェルプレートにおける所望の密度で、細胞を優先します。通常我々平板培地で最初のウェルの各項目を入力して、メディアの500μLの最終的な音量を得るために細胞懸濁液を追加することにより、10細胞/μL。 </li><li>それはあなたが7日後に発生する球をカウントするまで移動されない37℃CO 2インキュベーターでプレートを置きます。 </li></ol> 3。網膜幹細胞を単離するの結果<ol><li>この手順が正常に実行されると、解剖毛様体上皮細胞は低密度（図1）で解離し、メッキされた後、次のようになります。 </li><li>培養中の7日後、生じた網膜幹細胞の球がカウントされます。球体は、直径および幹細胞由来の球としてカウントされるフリーフローティングでは75μm以上にしてください。 （図2）。ただし、一部の細胞は大きさの基準を満たしていないと幹細胞由来の球としてカウントされず限られた増殖とフォームの回転楕円体を持つことになります。このような回転楕円体の例は（図2）ここに示されています。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/2209/2209fig1.jpg" alt="図1" /> 図1。 <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="図2" /> 図2。

<ol>
	<li>Tropepe, V., Coles, B. L., Chiasson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+in+the+adult+mammalian+eye.">Retinal stem cells in the adult mammalian eye.</a> Science. 287, 2032-2036 (2000).</li><li>Ahmad, I., Tang, L., Pham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+progenitors+in+the+adult+mammalian+eye.">Identification of neural progenitors in the adult mammalian eye.</a> Biochem Biophys Res Commun. 270, 517-521 (2000).</li><li>Coles, B. L., Angenieux, B., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+isolation+and+the+characterization+of+human+retinal+stem+cells.">Facile isolation and the characterization of human retinal stem cells.</a> Proc Natl Acad Sci U S A. 101, 15772-15777 (2004).</li><li>Cicero, S. A., Johnson, D., Reyntjens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proc Natl Acad Sci U S A. 106, 6685-6690 (2009).</li><li>Lamba, D., Karl, M., Reh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+cell+replacement:+a+view+from+the+eye.">Neural regeneration and cell replacement: a view from the eye.</a> Cell Stem Cell. 2 (6), 538-549 (2008).</li><li>Coles, B. L., Horsford, D. J., McInnes, R. R., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+retinal+progenitor+cells+leads+to+an+increase+in+the+retinal+stem+cell+population+in+vivo.">Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo.</a> Eur J Neurosci. 23, 75-82 (2006).</li><li>Ang&#233;nieux, B., Schorderet, F. D., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+is+a+neuronal+differentiation+factor+for+retinal+stem+cells+in+vitro.">Epidermal growth factor is a neuronal differentiation factor for retinal stem cells in vitro.</a> Stem Cells. 24 (3), 696-706 (2006).</li><li>Xu, S., Sunderland, M. E., Coles, B. 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+proliferation+and+expansion+of+retinal+stem+cells+require+functional+Pax6.">The proliferation and expansion of retinal stem cells require functional Pax6.</a> Dev Biol. 304, 713-721 (2007).</li><li>Canola, K., Ang&#233;nieux, B., Tekaya, M., Quiambao, A., Naash, M. I., Munier, F. L., Schorderet, D. 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利害の衝突は宣言されません。

このプロトコルは1-3マウスの眼の毛様体上皮から網膜幹細胞を分離する方法について説明します。毛様体上皮の細片を単離するための方法論は、しかしながら、単一細胞懸濁液を達成するための酵素と方法論は、このプロトコルに最適化されている、変えることができます。それは、これらは各々の眼から単離することができる球の総数にマイナスの影響を持っているように見えるので、分離されている毛様体上皮のストリップはRPEまたは角膜を大量に含まれていないことも重要です。さらに、我々は、もともとbrainニューロスフェアを13日に策定され、その作成 ​​することを無血清培地は、網膜幹細胞の球を成長させるための理想的です。一つは、また、それらが成長している球が一緒に14を集計しないように播種し、インキュベータ内に配置された後、細胞に障害が発生していないことを確認する必要があります。 網膜幹細胞がクローン性の球を形成した後、それらは眼あたりの幹細胞の見込み数を得るためにカウントすることができます。球体は、その後、単一細胞に解離さと把握自己再生能力に継代または成長因子および/またはタンパク質の異なる組み合わせを使用して神経網膜とRPEの異なる細胞型に分化することができます。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
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<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
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<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
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<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
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<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
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<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
</tr>		</tbody>
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isolation of retinal stem cells from the mouse eye

成体マウスの網膜幹細胞（RSC）は、哺乳類の目1,2,3の毛様体上皮（CE）に見られる珍しい静止細胞です。 CEは、非色素内側と色素外側の細胞層から構成され、そしてCEからの単一色素細胞から生じるクローンRSCコロニーはすべて形成するために区別することができます両方の色素と非色素細胞で構成されています神経網膜とRPEの細胞型。球内のすべての細胞はすべて、少なくともいくつかの顔料4を含むかどうかについてのいくつかの論争があり、しかし細胞はまだ1月3日神経網膜内に存在する異なる種類の細胞を形成することができる。このような両生類や魚類などのいくつかの種においては、彼らの目はしかし、怪我の5の後の再生が可能です。哺乳類の目にはそのような再生特性を示していない。我々は、in vivoで幹細胞を識別し、目のけがの修復物理的または遺伝的モデルを助けるために、細胞の潜在的なソースとして使用しているだけでなく、さらに損傷後、6-8静止哺乳類の網膜幹細胞を維持するメカニズムを理解しよう9月12日移植による。ここでは、クローン網膜幹細胞の球体を形成するために、マウスの眼から毛様体上皮細胞を分離し、文化の中で、それらを成長させる方法について説明します。 in vivoで幹細胞の既知のマーカーが存在しないため、これらの球はプロスペクティブに眼の毛様体上皮内に幹細胞集団を識別するための唯一の知られている方法です。

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

このビデオでは、我々はマウスの眼の毛様体上皮から網膜幹細胞を分離し、クローン網膜球体を形成する文化の中で、それらを成長させる方法を紹介します。分離されている球は、幹細胞の枢機卿性質を有している：自己再生および多能。

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-retinal-stem-cells-from-the-mouse-eye

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JoVE Journal

Biology

24.7K ビュー.  University of Toronto. このビデオでは、我々はマウスの眼の毛様体上皮から網膜幹細胞を分離し、クローン網膜球体を形成する文化の中で、それらを成長させる方法を紹介します。分離されている球は、幹細胞の枢機卿性質を有している：自己再生および多能。

ビデオ: Isolation of Retinal Stem Cells from the Mouse Eye

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

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 ...

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 ...

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 ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

マウス網膜色素上皮からの一次電池文化

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

ブタ網膜色素上皮細胞の初代培養

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

初代マウス網膜色素上皮細胞の単離

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...