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

这个项目是一个莱昂J.塔尔从加州再生医学研究所的种子批。托比亚斯亨宁是由德国研究协会（DFG，何4578/1-2）津贴一个研究。我们要感谢他对人类胚胎干细胞培养的意见的华尼托Meneses。

间质干细胞的标记与荧光染料<ol><li>要开始标签间质干细胞的过程，trypsinize和细胞计数，以获得与一个细胞的定义暂停。 </li><li>的孵化器中取出细胞，吸出含有被标记的细胞培养瓶旧媒体</li><li> 10毫升的Mg / Ca无PBS清洗细胞。吸出的PBS。镁和钙会抑制胰蛋白酶，因此我们用PBS没有这些。 </li><li>加入预热的0.05％胰蛋白酶，为T75烧瓶中，我们使用5毫升。确保覆盖整个表面的容量瓶。 </li><li>在37 ° C的培养箱孵育约5分钟。 </li><li>在显微镜下确认支队。如果细胞仍然坚持到烧瓶中，点击了几次，再等一会儿，直到胰蛋白酶是成功。 </li><li>现在，它是必要的胰蛋白酶中加入含10％FCS的媒体。我们使用等量的媒体有胰蛋白酶。 </li><li>移液器上下几次，以确保所有的细胞重悬在媒体。 </li><li>转移到无菌15毫升上限聚丙烯管的电池解决方案。 </li><li>在400 RCF离心5分钟。 </li><li>确保不打扰细胞沉淀，吸出上清液。 </li><li>在DMEM重悬细胞，并进行细胞计数。 </li><li>暂停细胞标记在密度为1 × 10 ^每毫升6在无血清培养基（DMEM）。 </li><li>这是所有的细胞悬液的制备。现在，它已经准备好开始的标签。 </li><li>首先，添加5μLDID造影剂每毫升细胞悬液。 </li><li>然后，轻轻吹打混合的解决方案。 </li><li>孵育标签解决方案，在37 ° C时20分钟在6孔盘低附着的细胞。 </li><li>一旦完成简单的孵化，这是需要转移的细胞溶液15毫升的上限聚丙烯管。 </li><li>在400 RCF离心5分钟。 </li><li>吸出的标签介质，确保不干扰细胞沉淀。 </li><li>用PBS清洗细胞。移液器的细胞向上和向下，一定要打破细胞沉淀。 </li><li>重复两次后两个步骤，所以共3个洗涤步骤。 </li><li>计数细胞，并进行台盼蓝测试，以确定细胞活力。这些都是标准的细胞培养协议，我们不会在这里解释。 </li><li>现在已经准备好你的细胞在光学成像仪成像。 </li></ol>人类胚胎干细胞标记的荧光染料ICG <ol><li>要开始标签人类胚胎干细胞的过程，准备吲哚青绿造影剂。转代理鱼精蛋白混合。 </li><li>测量出1mg的吲哚青绿粉。在100μL二甲基亚砜（DMSO）溶解ICG的粉末。 </li><li>的混合物中加入400μL贝科修改老鹰培养基（DMEM + 10％胎牛血清）媒体和动摇。在终浓度为2mg/ml这个结果吲哚青绿。 </li><li>添加转代理鱼精蛋白。鱼精蛋白作为造影剂穿梭的行为，因此，它得到更有效地进入细胞。 </li><li>混合5μL硫酸鱼精蛋白，这是提供在浓度为10mg/ml，用300μLICG和300μL无血清贝科的修改老鹰中等，。 </li><li>轻轻摇动5分钟，新的转染的解决方案，让复杂的形式。 </li><li>标签解决方案已准备就绪。 </li><li>从人类胚胎干细胞10mm的培养皿中吸出旧媒体。 </li><li>加入预热的无血清DMEM5毫升。 </li><li>添加细胞先前准备的鱼精蛋白/ ICG的解决方案。开始由放置在一个孵化器在37菜1小时的潜伏期° C。 </li><li>孵化完成后，从孵化器中取出的菜，吸出的标签解决方案。 </li><li>冲洗菜用5 mL PBS清洗细胞。 </li><li>吸出PBS和更换与0.25％胰蛋白酶5毫升。菜在37℃5分钟，使胰蛋白酶发生。它有助于在一段时间的菜一点点每一次握手。 </li><li>轻轻吸液管向上和向下，打破剩余的殖民地。 </li><li>胰蛋白酶中加入等量的KSR菜。 </li><li>细胞溶液转移到15毫升管，离心5分钟，在400 RCF的解决方案。 </li><li>重悬细胞，在全媒体。 </li><li>如果仍然是在这一点上的团块，trypsinize离心一次。 </li><li>一个丛免费的电池解决方案实现后，吸出旧媒体和悬浮颗粒在10ml预热的全ESC媒体。 </li><li>在这一点上，它是必要的，从人类胚胎干细胞的小鼠饲养层细胞分开。这样做是为了确保以后，只有成像的干细胞发生。 </li><li>对于这一点，转移日E细胞明胶涂层10 cm培养皿的解决方案。 </li><li>放入37℃培养箱菜，让它坐了45分钟。在这段时间内，确保不打扰的菜。现在，进料器将坚持以菜和干细胞不会。 </li><li>转出的培养皿中的解决方案。现在我们有人类胚胎干细胞的一个标记的单细胞的解决方案。 </li><li>这些细胞现在可以计数和台盼蓝测试，可以对它们执行。 </li><li>细胞进行成像准备！ </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是一个相对较新的成像技术，荧光检测的基础上。 Oi是示踪剂成像技术的敏感，但不与任何辐射暴露关联。其他投资提供跟踪细胞的非侵入性的和重复的一个有效手段，从而提供了了解细胞迁移到目标站点。该技术的一个主要限制是有限的荧光探针在体内的组织渗透。因此，在深部组织的示踪积累，多约5-10厘米的皮肤表面，可能不会被发现。目前临床上的应用是有限的肤浅的技术，如内镜，心血管疾病和视网膜成像（Funovics等，2003），但将继续扩大这方面的研究领域，通过跟踪体内细胞提供了广阔的可能性的认可。

<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

光学成像（OI），是一个方便，快捷，廉价的新的干细胞治疗的体内监测的工具。该技术是基于体内的干细胞用荧光染料，随后静脉注射标记的细胞，在特定的靶器官或病理积累的可视化标签。所提出的技术演示我们如何由简单的孵化与亲脂性荧光染料标签人类间质干细胞（HMSC）（C67H103CIN2O3S），我们如何标签人类胚胎干细胞（胚胎干细胞），美国FDA批准的荧光染料的吲哚菁绿（ICG）。吸收机制是通过坚持和推广整个磷脂细胞膜双层lypophilic染料。通常标记效率提高，如果细胞培养，而不是在含血清的介质中孵化的无血清培养基中的染料。此外，除了转染剂硫酸鱼精蛋白显着提高造影剂的吸收。

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

与非侵入性检测荧光染料标记的干细胞与光学成像

该视频显示技术，人类胚胎干细胞和骨髓基质干细胞与荧光染料标签。这种技术可以用于移植的干细胞在体内追踪与光学成像和荧光显微镜观察组织病理学的相关性。

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 次观看.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. 该视频显示技术，人类胚胎干细胞和骨髓基质干细胞与荧光染料标签。这种技术可以用于移植的干细胞在体内追踪与光学成像和荧光显微镜观察组织病理学的相关性。

视频: 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.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

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

科研

发育生物学

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;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

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.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

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.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

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.

从3维球体培养中分离出类似干细胞的细胞

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

癌症研究

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.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

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

神经科学

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.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

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.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

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.

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

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.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

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