This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

安定SOX2規制地域レポーター構築物を含むレンチウイルスで形質導入されたOVCAR-3ヒト卵巣癌細胞の1世代<ol><li> 10,21が説明されているようにSOX2調節領域を認識レポーター構築とHEK 293T-パッケージング細胞株をトランスフェクションすることによってレンチウイルス粒子を生成します。 注：レポーター構築物はさらに、先にtdTomatoの蛍光タンパク質のProteoTunerシステムシールドシステムの不安定化ドメインを含む。 Shield1を、それにより蛍光タンパク質22を分解するためのプロテアソームを防止不安定化ドメインに結合する。 </li><li> 24時間の期間にわたって、レンチウイルス粒子を有するOVCAR-3細胞を形質導入する。その後、ウイル​​ス上清を除去し、リン酸緩衝生理食塩水（PBS）、完全培地中で培養した（RPMI、10％FBS、100 U / mlのペニシリン、100μg/ mlのストレプトマイシン）で細胞を洗浄する。 </li><li> 48時間後、10μg/ mlののプーロマイシンを培養物に添加し、適切に形質導入​​された細胞の選択を可能にするために5日間維持した。 </li></ol>細胞選別、めっきの作製<ol><li>前細胞選別1,000希釈24時間：1でのShield1を追加します。ネガティブコントロールとしてのShield1処理（ 図1）することなく、安定的に形質導入されたOVCAR-3細胞を使用してください。フラスコから培地を吸引除去し、3分間、0.05％トリプシン-EDTAを用いて細胞を1×PBSで細胞を洗浄し、トリプシン処理。 </li><li> 5分間 - （25°C 15）完全培地を使用してトリプシンを停止し（上記参照）、細胞数をカウントし、室温で300×gで遠心分離細胞。 </li><li> 1mlの滅菌PBS - 0.5に慎重に上清を再懸濁細胞をデカントします。 </li><li>単一細胞懸濁液を得るために、40μmのセルストレーナーキャップフィルタを使用する。 </li><li>ミリリットルあたり5万個の細胞に細胞数を調整します。 </li><li>培地を100μlの球で超低付着の96ウェルプレートを準備し（MEGMは、増殖因子、サイトカイン、およびサプリメントを補充B-27、ヘパリンナトリウム;またはDMEM / F12）は、 表1を参照して、1％メチルセルロースの添加の有無にかかわらず、成長因子、サイトカイン、およびサプリメント、B-27、ヘパリンナトリウムを補充した。必要に応じて可能な汚染のリスクを最小限にするために100 U / mlのペニシリンおよび100μg/ mlのストレプトマイシンの濃度で培地に抗生物質を加える。 </li><li>ソートRFP +上から調製した96ウェルプレートにRFP-細胞を、ウェルあたり1細胞（単球、細胞ベースアッセイ）及びウェル当たり100細胞（多細胞ベースの球アッセイ）。 100μノズル、シース圧力20 PSI、および歩留まりマスク0、純度マスク32、位相マスク16：シングルセルモード、ソートセットアップを使用して（ 材料を参照）ソートで市販のセルソーターを実行します。 </li><li>多細胞ベースのアッセイのために（顕微鏡的（単一の細胞ベースのアッセイのために）細胞を含むウェルをスコアによって個々のウェル中の細胞数を計数することにより、めっき効率を評価する; 図2 </stronG&gt;）。 </li><li> 37℃、5％CO 2で（組成についてステップ2.6参照）培地球における標準条件下で細胞をインキュベートする。毎日のbFGF（20ng / ml）およびEGF（20ng / mlの）を補完する。 </li><li> 1週間後、統合されたレポーターシステムからの蛍光シグナルを検出するための4倍または10倍の倍率蛍光顕微鏡で標準的な顕微鏡を使用して、新たな腫瘍球の数を数える。 「大」球としては100μmを超える直径の球をカウントし、直径50球- &quot;小さな&quot;球（ 図3）としては100μm。あなたが本当の球をカウントし、クラスターをセルではないことを確認してください。 注：単一のセルベースアッセイでは、球形成は文化の10（7対）日後に顕微鏡的に得点する方が簡単です。 </li><li> RFPの+中球形成細胞の割合を計算し、それぞれの単一細胞ベースのアッセイ（各個々の実験のために1枚の96ウェルプレート）または多細胞学士でRFP-細胞ショーらによって提示されたsed球アッセイ（各個々の実験用のウェル）。16 注：細胞を形成する球の割合（％）=（球の数）/（播種した細胞数）×100 </li></ol>球の3連続継代<ol><li> RTで10分間300×gで、適切な滅菌チューブと遠心分離機における各ウェルのコンテンツを配置します。マルチセルベース球アッセイのために、1つのウェルから球を一緒に集める。 PBSおよび2分より長い遠心分離機で5回 - よく3を洗う。単一細胞ベースの球アッセイのために、個々の球を集める。起因する細胞の数が少ないために、遠心分離および洗浄工程では1.5 mlチューブを使用しています。 </li><li> 0.05％トリプシン-EDTAの200μlの上清と再懸濁ペレットを削除します。 </li><li>最適な細胞分離を達成するために、ソフトシェーカーで5分間、37℃で細胞懸濁液をインキュベートする。細胞死率を下げるためにあなたの細胞株について、トリプシン処理時間を最適化：なし大型た場合球は穏やかに100μlのピペットチップを用いて可視粉砕物である。ケースで大球は、まだ存在している別の3分のトリプシンでインキュベートした後、トリチュステップに進みます。シングルセルベースアッセイの場合、トリプシン処理工程中の生きた細胞の最適な細胞収量のための時間を最適化するようにしてください。 </li><li>トリプシンを不活性化するために、単一細胞アッセイの場合には、10分間、500μlのに追加の2分間の遠心分離を300×gで遠心分離し、完全培地を加える。 </li><li>球培地に注意深く上清および再懸濁細胞を除去する。 </li><li>単一細胞懸濁液を得るために、40μmのセルストレーナーキャップフィルタを使用する。 </li><li> RFP +および多細胞bassedアッセイにおいてRFP-細胞を用いる場合には、フローサイトメーター分析を介して十分に再懸濁した後、各蛍光細胞の割合を評価する。 </li><li>上記で詳述したように調製した単一細胞の連続再プレーティングアッセイ、シードウェルあたり1細胞の新しい超低付着の96ウェルプレートに用。 1個々の球体からは、約20個々のウェルをシード。マルチセルベースの​​プライマリ球の再プレーティングアッセイのために、種子細胞を新しい96ウェルプレートに一次球の1つのウェルから得られ、顕微鏡法によって細胞数を翌日カウント。 </li><li> ステップ2.11で説明した式を用いた二球アッセイにおける球形成細胞の割合を評価する。 </li></ol> 4.結果解析<ol><li>独立した三連で行われた実験の結果を分析し、統計分析のために、正規分布の値とそれ以外のマン - ホイットニー·テストを分析するために、両側スチューデントのt検定を使用しています。 </li></ol>

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F., et al. <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+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

球培養物は、癌幹細胞は、潜在的なアッセイおよびヒト腫瘍細胞15,25,26広範囲の幹様細胞を濃縮するために広く用いられている方法である。これらの培養条件下で、自己複製能を欠く癌細胞は分化し、最終的に細胞死を受けることが予想される。彼らは当初、特に第一のアッセイであっても腫瘍球を細胞クラスターを形成するか、またはかもしれないが、それらは、自己複製の特性の...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

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			<td>Lonza</td>
			<td>CC-3151</td>
			<td></td>
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			<td height="21" style="height:21px;">Insulin</td>
			<td>Lonza</td>
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			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
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			<td>Sigma</td>
			<td>E9644</td>
			<td>end concentration: 20 ng/ml</td>
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			<td>PeproTech</td>
			<td>100-18B</td>
			<td>end concentration: 20 ng/ml</td>
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			<td>Invitrogen/ Gibco</td>
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			<td>end concentration: 1x</td>
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			<td height="21" style="height:21px;">Heparin-Natrium-25000 IE</td>
			<td>Ratiopharm</td>
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			<td>dilution 1:1000</td>
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			<td>Gibco</td>
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			<td height="21" style="height:21px;">FCS&#160;</td>
			<td>Gibco</td>
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			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
			<td></td>
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			<td height="21" style="height:21px;">Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
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			<td height="21" style="height:21px;">Shield1&#160;</td>
			<td>Clontech</td>
			<td style="width:119px;">632189</td>
			<td>dilution 1:1000</td>
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			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl cellulose</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M0387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td>applichem</td>
			<td style="width:119px;">A2856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cell sorter&#160;</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>Aria III cell sorter&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS analyser</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>accuri c6 flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:112px;">Olympus&#160;</td>
			<td style="width:119px;"></td>
			<td>IX50 Osiris</td>
		</tr>
	</tbody>
</table>

evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

従来の球アッセイにおいて、RFP + OVCAR-3細胞の約40％対RFP-細胞の20％が一次球アッセイにおける個々の腫瘍球（ 図4A）を生じた。また、RFP +細胞によって形成球体はRFP-細胞によって形成されたものよりもサイズが大きかった。 単一細胞に基づくアッセイでプレーティングすると、RFP +細胞はまた、上記の結果を確認し、RFP-細胞より球を形成した。しかし、多細...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

マルチとシングルセルベース球アッセイを用いてヒト卵巣癌細胞における幹細胞特性の評価

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

Research

JoVE Journal

Medicine

9.5K ビュー.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

ビデオ: Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

医学

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

区分するマイクロ流体デバイスとライブセルイメージングを使用した癌幹細胞の移行の評価

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

ソート幹細胞アッセイおよびフローの原発性脳腫瘍組織の処理

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

携帯誘電分光法による特発性側弯症の予後予測のための細胞ベースのアッセイプロトコール

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

用RNAscope<em&gt;その場で</em&gt;ヘッドの転写的に活性なヒトパピローマウイルスの検出および頸部扁平上皮癌

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人間のてんかん術後皮質組織の多電極アレイ録音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

ヒト細胞株から化学療法抵抗性の卵巣がん幹細胞の濃縮

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...