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

Bu proje için California Rejeneratif Tıp Enstitüsü bir Leon J. Thal TOHUM hibe ile desteklendi. Tobias Henning, Alman Araştırma Birliği (DFG, HE 4578/1-2) nakit Araştırma tarafından finanse edildi. Biz insan embriyonik kök hücre kültürü onun tavsiye Juanito Meneses minnetle kabul etmek istiyorum.

Floresan boya ile mezenkimal kök hücrelerin Etiketleme DID <ol><li> Mezenkimal kök hücreleri etiketleme prosedürü başlatmak için, trypsinize ve hücrelerin belirli bir sayı ile bir süspansiyon elde etmek için hücre sayımı. </li><li> Kuluçka hücreleri ve etiketli hücreleri içeren şişeler eski medya aspirat </li><li> Mg / Ca-free PBS 10 ml hücreleri yıkayın. PBS aspire. Mg ve Ca tripsin inhibe eder, bu nedenle biz bu olmadan PBS kullanabilirsiniz. </li><li> 5ml kullanan T75 şişesi için, önceden ısıtılmış% 0.05 tripsin ekleyin. Balonun tüm yüzeyi kaplı olduğundan emin olun. </li><li> Yaklaşık 5 dakika için 37 ° C inkübatör &#39;da inkübe edin. </li><li> Mikroskop altında dekolmanı onaylayın. Hücreleri hala balona bağlı ise, birkaç kez dokunun ve trypsinization başarılı oluncaya kadar biraz daha bekleyin. </li><li> Şimdi,% 10 FCS içeren medya ekleyerek tripsin nötralize etmek için gerekli. Tripsin var olduğu gibi bir ortam eşit miktarda kullanın. </li><li> Olduğunu tüm hücreleri medyada tekrar askıya sağlamak için birkaç kez aşağı yukarı Pipet. </li><li> Hücre çözümü 15ml steril kapaklı polipropilen tüp aktarın. </li><li> 5 dakika boyunca 400 RCF santrifüjleyin. </li><li> Hücre pelletini rahatsız etmeyecek şekilde sağlanması süpernatantı aspire. </li><li> DMEM hücrelerin yeniden süspanse edin ve hücre sayımı ile devam edin. </li><li> ^ 6 ml serum serbest kültür vasatı (DMEM) başına 1x10 yoğunluğu etiketlenmesine hücreleri askıya alınması. </li><li> Bu hücre süspansiyonu tüm hazırlık oldu. Şimdi, etiketleme başlamak için hazır. </li><li> İlk olarak, hücre süspansiyonu ml başına kontrast madde 5 mcL ekleyin. </li><li> Sonra, yavaşça pipetleme çözüm karıştırın. </li><li> Etiketleme çözümü 37 ° C de 20 dakika 6-iyi bir düşük-attachment çanak hücreleri inkübe edin. </li><li> Basit inkübasyon tamamlandıktan sonra, 15 ml kapaklı polipropilen tüp hücre çözümü aktarmak için gereklidir. </li><li> 5 dakika boyunca 400 RCF santrifüjleyin. </li><li> Hücre pelletini rahatsız sağlanması, etiketleme orta aspire edin. </li><li> Hücreler PBS ile yıkayın. Hücreler yukarı ve aşağı Pipet, hücre pelletini break up emin olun. </li><li> Son iki adımı iki kez daha tekrarlayın, bu nedenle bir toplam 3 yıkama adımları var. </li><li> Hücre sayımı ve hücre canlılığını belirlemek için bir Tripan mavi testi gerçekleştirin. Bunlar, standart hücre kültürü protokolleri burada anlatmayacağım olduğunu. </li><li> Hücreler artık optik görüntüleyici görüntülü hazır. </li></ol> Floresan boya ICG ile insan embriyonik kök hücreleri Etiketleme <ol><li> Insan embriyonik kök hücreleri etiketleme prosedürü başlatmak için, kontrast madde Yeşil İndosiyanin hazırlamak. Transfeksiyon ajan Protamin ile karıştırın. </li><li> İndosiyanin Yeşil toz 1mg ölçün. Dimetil sülfoksit (DMSO) 100 mcL ICG tozu çözülür. </li><li> Dulbecco&#39;nun Modifiye Eagles Medium (DMEM +% 10 Fötal Buzağı Serumu) medya 400 mcL karışıma ekleyin ve iyice sallayın. Bu sonuç, 2mg/ml son bir konsantrasyon Yeşil İndosiyanin. </li><li> Transfeksiyon ajan Protamin ekleyin. Kontrast madde için bir servis olarak protamin eylemler, bu nedenle daha verimli bir şekilde hücre içine alır. </li><li> 300 mcL ICG ve 300 mcL serum serbest Dulbecco&#39;nun Modifiye Eagles Orta 10mg/ml bir konsantrasyon sağlanır 5 mcL protamin sülfat, karıştırın. </li><li> Formu karmaşık izin vermek için 5 dakika için yeni transfeksiyon çözüm yavaşça sallayın. </li><li> Etiketleme çözümü hazırdır. </li><li> HESC 10mm Petri kabı eski medya aspire. </li><li> Önceden ısıtılmış serum serbest DMEM 5ml ekle. </li><li> Hücreleri için önceden hazırlanmış Protamin / ICG solüsyonu ekleyin. 37 bir kuluçka makinesi, bulaşık koyarak 1 saat inkübasyon Başlat ° C </li><li> Inkübasyon tamamlandıktan sonra, inkübatör çanak kaldırmak ve etiketleme çözümü aspirat. </li><li> 5 ml PBS ile bulaşık durulama hücrelerin yıkayın. </li><li> PBS aspire ve% 0.25 tripsin, 5ml ile değiştirin. 37 ° C&#39;de 5 dakika trypsinization meydana izin çanak inkübe edin. Bu biraz her seferinde bir süre içinde çanak sallamak için yardımcı olur. </li><li> Yavaşça kalan koloniler break up, pipet ve aşağı yukarı. </li><li> KSR bir eşit miktarda çanak ekleyerek tripsin nötralize. </li><li> 15ml tüp hücre çözümü aktarın ve 5 dakika boyunca 400 RCF çözüm santrifüj. </li><li> Tam medya hücrelerin yeniden süspanse edin. </li><li> Hala bu noktada az kümeleri varsa, trypsinize ve tekrar santrifüj. </li><li> Bir tutam ücretsiz cep çözüm elde edildikten sonra, eski medya aspirat ve önceden ısıtılmış tam ESC medya 10ml pelet tekrar süspansiyon haline getirin. </li><li> Bu noktada, hESC fare besleyici hücrelerini ayırmak için gereklidir. Bu emin olmak için yapılır, daha sonra, sadece görüntüleme kök hücreler oluşur. </li><li> Bunun için, inci transferijelatin kaplı 10 cm çanak e hücre çözümü. </li><li> Çanak 37 ° C inkübatör içine koyun ve 45 dakika bekletin. Bu süre zarfında, çanak rahatsız emin olun. Şimdi, çanak besleyiciler uyacaktır ve kök hücrelerin olmaz. </li><li> Petri kabı çözüm dışarı aktarın. ve şimdi insan embriyonik kök hücre etiketli bir tek hücre çözümü var. </li><li> Hücreleri sayılabilir ve Tripan mavi test yapılabilir. </li><li> Hücreleri yansıması için hazırız! </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 floresan algılama göre, görece yeni bir görüntüleme tekniğidir. OI radyotracer tabanlı görüntüleme teknikleri gibi hassas, ancak herhangi bir ışınlama maruz kalma ile ilişkili değildir. OI, böylece hedef site hücre göçü içgörü sağlayan non-invazif ve tekrarlayan izleme hücreleri etkili bir araç sağlar. Tekniğin önemli bir sınırlama in vivo floresan probları sınırlı doku penetrasyonu. Böylece, cilt yüzeyinden 5-10 cm daha derin dokularda bir izleme birikimi, tespit olmayabilir. Mevcut klinik uygulamal...

<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

Optik görüntüleme (OI), yeni kök hücre tabanlı tedaviler in vivo olarak izlenmesi için kolay, hızlı ve ucuz bir araçtır. Teknik floresan boya etiketli hücreleri belirli hedef organları veya patolojiler kendi birikimi ve görselleştirme sonraki intravenöz enjeksiyon ile kök hücrelerinin ex vivo etiketleme dayanmaktadır. Sunulan tekniği (C67H103CIN2O3S) ve biz insan embriyonik kök hücreleri (hESC) etiket nasıl FDA floresan boya İndosiyanin Yeşil (ICG) onaylı lipofilik floresan boya ile basit inkübasyon insan mezenkimal kök hücreleri (hMSC) etiket nasıl gösterir. Fosfolipid hücre zarı bilayeri genelinde lypophilic boya bağlılık ve difüzyon yoluyla alımı mekanizması. Hücreler serum içeren medya inkübasyon karşı serum serbest medya boya ile inkübe edilir etiketleme verimliliği artırıldı. Ayrıca, transfeksiyon ajan Protamin Sülfat ek kontrast madde alımını önemli ölçüde artırır.

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

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Bu video floresan boya ile insan embriyonik kök hücreler ve mezenkimal kök hücreler etiketleme teknikleri gösterir. Bu teknik, in vivo olarak bir optik görüntüleme ile nakledilen kök hücrelerin izleme ve floresan mikroskobu ile histopatolojik korelasyon kullanılabilir.

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

Research

JoVE Journal

Biology

13.3K Görüntüleme.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. Bu video floresan boya ile insan embriyonik kök hücreler ve mezenkimal kök hücreler etiketleme teknikleri gösterir. Bu teknik, in vivo olarak bir optik görüntüleme ile nakledilen kök hücrelerin izleme ve floresan mikroskobu ile histopatolojik korelasyon kullanılabilir.

Video: 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.

Çok renkli Floresan Reporter Fare kullanma Kornea Hücre Lineage İzleme Bir Yöntem

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

Gelişim Biyolojisi

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.

Analiz<em&gt; İn Vivo</em&gt; Hücre Göç Zebra balığı Embriyolar hücre nakli ve Time-lapse Görüntüleme kullanılarak

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.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

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

Biyoloji

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 ve in vivo Biyoparlaklık Reporter Gen Görüntüleme İnsan Embriyonik Kök Hücreleri

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 Boyutlu Spheroid Kültürlerden Kök Benzeri Hücrelerin İzolasyon

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

Kanser Araştırmaları

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.

Fareler ve Merkezi Sinir Sisteminde Kök Hücre İmplantasyonu Multimodal Görüntüleme

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

Nörobilim

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.

Vivo İnsan yağ elde edilen Mezenkimal kök hücrelerin sıçan diz osteoartrit modelinde floresan lipofilik membran boya ile izleme

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.

Meme Tümörü Mikroçevresinin İntravital Görüntülemesi için Etiketsiz Bir Segmentasyon Yaklaşımı

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.

Fare Retinal Sirkülasyonunda Akış Dinamikleri Çalışmak İçin Eritrositler ve Lökositlerin Floresan Boya Etiketlenmesi

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

Tıp

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.

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

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