Name: labeling stem cells with fluorescent dyes for non-invasive detection with optical imaging
Uploaded: 2008-04-02T18:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging at JoVE.com

このプロジェクトは、再生医療のためのカリフォルニア工科大学からレオンJ.タールのSEEDの助成金によってサポートされていました。トビアスヘニングは、ドイツ研究協会（DFG、HE 4578/1-2）から奨学金研究によって資金を供給された。我々は感謝してヒト胚性幹細胞の培養上彼のアドバイスをファニートのMenesesを確認応答したい。

蛍光色素との間葉系幹細胞のラベリングは、DID <ol><li>間葉系幹細胞を標識するための手順を開始するには、トリプシン処理および細胞の定義の数と、懸濁液を得るために細胞を数える。 </li><li>インキュベーターから細胞を取り出し、ラベルを付ける細胞を含むフラスコから古い培地を吸引除去</li><li>のMg / Caのフリー10mlのPBSで細胞を洗浄する。 PBSを吸引除去する。 MgおよびCaは、トリプシンを阻害する、従って私達はこれらなしでPBSを使用してください。 </li><li>我々は5mlを使用するT75フラスコのために、予め温めておいた0.05％トリプシンを追加。フラスコの表面全体が覆われていることを確認してください。 </li><li>約5分間37インキュベーターで℃でインキュベートする。 </li><li>顕微鏡下で剥離を確認してください。細胞がまだフラスコに付着した場合、それを数回タップし、トリプシン処理が成功するまで少し長く待つ。 </li><li>今、それは10％FCSを含有する培地を加えることによってトリプシンを中和する必要があります。トリプシンがあるので、我々はメディアの同じ量を使用してください。 </li><li>すべての細胞は培地に再懸濁していることを確認するために数回上下にピペッティングし。 </li><li>滅菌15ミリリットルキャップポリプロピレンチューブに細胞溶液を移す。 </li><li> 5分間、400 rcfで遠心する。 </li><li>細胞ペレットを崩さないように確実に上清を吸引除去する。 </li><li> DMEMで細胞を再懸濁し、細胞数に進みます。 </li><li>無血清培地（DMEM）のmlあたり^ 6 × 10の密度で標識される細胞をサスペンド。 </li><li>これは、細胞懸濁液のすべての準備だった。今、それはラベルを開始する準備ができています。 </li><li>最初に、細胞懸濁液のmlあたり、DID造影剤を5μLを加える。 </li><li>その後、軽くピペッティングすることによってソリューションを混在させること。 </li><li> 37標識溶液℃で6 - ウェル低アタッチメント皿で20分間で細胞をインキュベートする。 </li><li>シンプルなインキュベーションが完了したら、それは15ミリリットルキャップポリプロピレンチューブに細胞溶液を転送する必要があります。 </li><li> 5分間、400 rcfで、それを遠心してください。 </li><li>細胞ペレットを乱さないように確実に、標識メディウムを吸引除去する。 </li><li> PBSで細胞を洗浄。細胞ペレットを分割することを確認し、上下のセルをピペッティング。 </li><li>後者の二つのステップをさらに2回繰り返し、3の洗浄工程の合計があるので。 </li><li>細胞をカウントし、細胞の生存を決定するためにトリパンブルーテストを実行します。これらは、我々がここで説明するつもりはないされている標準的な細胞培養のプロトコルです。 </li><li>あなたの細胞は、光イメージャで撮像される準備が整いました。 </li></ol>蛍光色素ICGとヒト胚性幹細胞の標識<ol><li>ヒト胚性幹細胞を標識するための手順を開始するには、インドシアニングリーン造影剤を​​準備する。トランスフェクション剤のプロタミンと混ぜる。 </li><li>インドシアニングリーン粉1mgのを測定します。ジメチルスルホキシド（DMSO）を100μLでICGの粉末を溶かし。 </li><li>混合するダルベッコ改変イーグル培地（DMEM + 10％ウシ胎児血清）メディア400μLを加え、よくそれを振る。インドシアニングリーンの2mg/mlの最終濃度でこれが結果。 </li><li>トランスフェクションエージェントのプロタミンを追加。造影剤のためのシャトルとしてプロタミン行為、それはより効率的に細胞内に取得できるように。 </li><li> 300μLICGと300μLの無血清ダルベッコ改変イーグル培地で、10mg/mlの濃度で供給される5μL硫酸プロタミンを、混ぜる。 </li><li>ゆっくりとフォームへの複雑なように5分のための新しいトランスフェクション溶液を振る。 </li><li>標識溶液が準備ができています。 </li><li> hESCの10ミリメートルのシャーレから、古いメディアを吸引除去する。 </li><li>予め温めておいた血清を含まないDMEM 5mlを追加します。 </li><li>細胞に予め用意されたICG /プロタミン溶液を加える。 37℃インキュベーターに皿を置くことによって1時間のインキュベーションを開始℃に</li><li>インキュベーションが完了したら、インキュベーターから皿を削除し、標識溶液を吸引除去する。 </li><li> 5mlのPBSで皿を洗浄して細胞を洗浄する。 </li><li> PBSを吸引除去し、0.25％トリプシン5mlのと交換してください。 ℃で5分間トリプシン処理を発生させるための37皿をインキュベートする。それは、たまに料理を少し振るのに役立ちます。 </li><li>静かに残りのコロニーを分割するために、上下にピペッティングし。 </li><li>皿にKSRの同量を追加してトリプシンを中和する。 </li><li> 15ミリリットルチューブに細胞溶液を移し、5分間で400 rcfで解決策を遠心する。 </li><li>完全なメディアで細胞を再懸濁します。 </li><li>この時点でも塊がある場合は、トリプシン処理し、再度遠心する。 </li><li>塊のない細胞溶液が達成されると、古いメディアを吸引除去し、予め温めておいたフルESCメディア10mlのでペレットを再懸濁します。 </li><li>この時点で、それはヒトES細胞からマウスのフィーダー細胞を分離する必要がある。これはことを保証するために行われ、後に、唯一のイメージング幹細胞が発生します。 </li><li>このためには、目を移すゼラチンコートした10cmディッシュへの電子の細胞溶液。 </li><li> 37℃のインキュベーターに料理を入れて、それが45分放置します。この時間の間に、料理を邪魔しないように注意してください。今、フィーダーは皿に付着し、幹細胞はしません。 </li><li>ペトリ皿の中から解決策を転送する。そして今、私たちはヒト胚性幹細胞の標識した単一細胞のソリューションを持っている。 </li><li>細胞は、今カウントすることができますし、トリパンブルー試験は、それらを実行することができます。 </li><li>細胞は、イメージを作成する準備が整いました！ </li></ol>

<ol>
	<li>Daldrup-Link, H. E., Rudelius, M., Metz, S., Piontek, G., Settles, M., Pichler, B., Heinzmann, U., Weinmann, H. J., Schlegel, J., Link, T. M., Rummeny, E. J., Oostendorp, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+tracking+with+Gadophrin-2+--+a+bifunctional+contrast+agent+for+MR+imaging,+optical+imaging+and+fluorescence+microscopy.">Stem cell tracking with Gadophrin-2 -- a bifunctional contrast agent for MR imaging, optical imaging and fluorescence microscopy.</a> Eur J Nucl Med Mol Imaging. 31, 1312-1321 (2004).</li><li>Simon, G. H., Daldrup-Link, H. E., Kau, J., Metz, S., Schlegel, J., Piontek, G., Saborowski, O., Demos, S., Duyster, J., Pichler, 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=Optical+imaging+of+experimental+arthritis+using+allogeneic+leukocytes+labeled+with+a+near-infrared+fluorescent+probe.">Optical imaging of experimental arthritis using allogeneic leukocytes labeled with a near-infrared fluorescent probe.</a> Eur J Nucl Med &amp; Mol Imaging. 33, 998-1006 (2006).</li><li>Sutton, E., Henning, T., Pichler, B., Bremer, C., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Tracking+with+Optical+imaging.">Cell Tracking with Optical imaging.</a> Eur Radiol. 2, 350-357 (2003).</li><li>Funovics, M. A., Alencar, H., Su, H. S., Khazaie, K., Weissleder, R., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+multichannel+near+infrared+endoscope+for+mouse+imaging.">Miniaturized multichannel near infrared endoscope for mouse imaging.</a> Mol Imaging. 2, 350-357 (2003).</li></ol>

OIは、蛍光の検出に基づいて、比較的新しいイメージング技術です。 OIは、放射性トレーサーベースのイメージング技術と同じくらい敏感ですが、任意の照射への暴露に関連付けられていない。 OIは、それによって標的部位への細胞移動への洞察を提供する、非侵襲的かつ反復的に追跡する細胞の効果的な手段を提供します。テクニックの一つの主要な制限は、in vivoでの蛍光プローブの限られた組織の浸透です。このように、皮膚の表面から約5-10 cmより多くの深部組織中のトレーサの集積は、検出されない場合があります。現在の臨床応用はこのような、内視鏡心血管および網膜イメージング（Funovicsら、2003）などの表面的なテクニックに限定されているが、研究のこのドメインは、生体内細胞追跡にによって提供される広大な可能性の認識を通して拡大していきます。

<table><tbody><tr><td>Indocyanine Green (IR-125)</td><td>Reagent</td><td>Fisher Scientific</td><td>AC41254-1000</td><td></td></tr><tr><td>DMSO</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D-2650</td><td></td></tr><tr><td>D-MEM High Glucose </td><td>Reagent</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>Gelatin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G1890</td><td>Dilute to final concentration of 0.1% in PBS</td></tr><tr><td>PBS, Mg and Ca free</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Trypsin-EDTA 0.05% </td><td>Reagent</td><td>Invitrogen</td><td>25300-120</td><td></td></tr><tr><td>Trypsin-EDTA 0.25% </td><td>Reagent</td><td>Invitrogen</td><td>25200-114</td><td></td></tr><tr><td>FCS, characterized</td><td>Reagent</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>Cell Strainer (40 &amp;mu;m Nylon)</td><td>Reagent</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>DiD</td><td>Reagent</td><td>Molecular Probes, Life Technologies</td><td>V-22887</td><td></td></tr><tr><td>Knockout DMEM</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>10829018</td><td></td></tr><tr><td>Knockout Serum Replacer</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>10828028</td><td></td></tr><tr><td>b-Mercapt&amp;#8211;thanol </td><td>Reagent</td><td>Sigma-Aldrich</td><td>7522</td><td></td></tr><tr><td>Non-essential Amino Acids </td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>11140050</td><td></td></tr><tr><td>FGF-2</td><td>Reagent</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>Glutamine 200mM </td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>25030081</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Protamine Sulfate</td><td>Reagent</td><td>American Pharmaceutical Partners</td><td></td><td></td></tr></tbody></table>

labeling stem cells with fluorescent dyes for non-invasive detection with optical imaging

光イメージング（OI）は、新しい幹細胞ベースの治療のin vivoモニタリングのための簡単​​、迅速かつ安価なツールです。技術は、蛍光色素、標識細胞と特定の標的臓器や病態におけるその蓄積の可視化のその後の静脈注射による幹細胞のex vivoでの標識に基づいています。紹介したテクニックは、我々は脂溶性蛍光色素を使って簡単なインキュベーションすることによりヒト間葉系幹細胞（hMSCの）ラベルを付ける方法を示していました（C67H103CIN2O3S）と我々はラベル付け方法FDAによるヒト胚性幹細胞（ヒトES細胞）蛍光色素はグリーン（ICG）をインドシアニン承認した。取り込み機構は、リン脂質の細胞膜の脂質二重層でlypophilic染料の付着と拡散を介してです。細胞を、血清含有培地でのインキュベーションとは対照的に無血清培地中の色素とインキュベートされている場合の標識効率は、通常、改善されています。さらに、トランスフェクション剤、硫酸プロタミンの添加は、造影剤の取り込みを大幅に向上させます。

Watch this Scientific Journal Video about Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging at JoVE.com

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

このビデオでは、蛍光色素を持つヒト胚性幹細胞と間葉系幹細胞のラベリングのテクニックを示しています。この手法は、生体内で光学イメージングを、蛍光顕微鏡による病理組織学的相関の移植された幹細胞の追跡に使用することができます。

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

labeling-stem-cells-with-fluorescent-dyes-for-non-invasive-detection

Universidad San Sebastian

Research

JoVE Journal

Biology

13.3K ビュー.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. このビデオでは、蛍光色素を持つヒト胚性幹細胞と間葉系幹細胞のラベリングのテクニックを示しています。この手法は、生体内で光学イメージングを、蛍光顕微鏡による病理組織学的相関の移植された幹細胞の追跡に使用することができます。

ビデオ: Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

マルチカラー蛍光レポーターマウスを用いた角膜細胞の系譜トレースする方法

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

発生生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;インビボ</emゼブラフィッシュ胚における細胞移植とタイムラプスイメージングを使用して&gt;細胞移動

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

生物学

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3次元スフェロイド培養からの幹様細胞の分離

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

がん研究

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

神経科学

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

生体内でひと脂肪由来間葉系幹細胞が脂溶性膜の蛍光色素をラット膝関節変形性関節症モデルでの追跡

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

マウス網膜循環における血流動態を調べるための赤血球および白血球の蛍光色素標識

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

医学

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...