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

이 프로젝트는 재생 의료에 대한 캘리포니아 연구소에서 레온 J. Thal 시드 부여에 의해 지원되었다. 토바 이어스 헤닝는 독일 연구 협회 (DFG, HE 4578/1-2)에서 급비 연구에 의해 투자되었다. 우리는 기꺼이 그 인간의 배아 줄기 세포의 문화에 대한 그의 조언 후아의 Meneses을 인정 싶어요.

형광 염료와 mesenchymal 줄기 세포의 라벨링 것은나요 <ol><li> mesenchymal 줄기 세포를 라벨에 대한 절차를 시작하려면 trypsinize 및 세포의 정의된 번호를 중지를 얻을 수있는 세포를 계산합니다. </li><li> 인큐베이터 밖으로 세포를 가지고 세포가 표시되는 포함된 flasks에서 이전 미디어를 기음 </li><li> MG / CA - 무료 PBS 10 ML있는 세포를 씻으십시오. PBS를 대기음. MG와 CA는 트립신을 억제 것이다 그러므로 우리는이없이는 PBS를 사용합니다. </li><li> 우리가 5ml를 사용하여 T75 플라스크에 대한 사전 예열 0.05 %의 트립신을 추가합니다. 술병의 전체 표면이 적용되었는지 확인하십시오. </li><li> 약 5 분 동안 37 ° C 배양기에서에 품어. </li><li> 현미경으로 초연을 확인합니다. 세포가 계속 술병을 준수하면, 몇 번 누르고 trypsinization이 성공 때까지 조금 더 기다려. </li><li> 이제 10 % FCS를 포함하는 미디어를 추가하여 트립신을 중화하는 것이 필요합니다. 트립신가로서 우리는 매체의 동일한 금액을 사용합니다. </li><li> 모든 세포가 언론에 다시 중단되는 것을 보장하기 위해 몇 번 위아래로 피펫합니다. </li><li> 멸균 15ml 덮인 폴리 프로필렌 튜브에 세포 솔루션을 전송합니다. </li><li> 5 분 400 RCF에서 원심 분리기. </li><li> 세포 펠렛을 방해하지 않도록 보장 뜨는을 대기음. </li><li> DMEM에있는 세포를 Resuspend 및 세포 개수와 함께 진행합니다. </li><li> 혈청이없는 배지 (DMEM)에 ML 당 ^ 6 1x10의 밀도에 표시되도록 세포를 일시 중지합니다. </li><li> 이것은 세포 현탁액의 모든 준비했습니다. 지금, 그것은 상표를 시작할 준비가 된 것입니다. </li><li> 첫째, 세포 현탁액의 ML 당나요 대비 에이전트의 5 μL를 추가합니다. </li><li> 그런 다음, 부드럽게 pipetting하여 솔루션을 섞는다. </li><li> 37 라벨 솔루션 ° 6 - 잘 낮은 첨부 접시에 20 분 C와 세포를 품어. </li><li> 간단한 배양이 완료되면, 그것은 15 ML 덮인 폴리 프로필렌 튜브에 세포 솔루션을 전송하는 것이 필요합니다. </li><li> 5 분 400 RCF에서 내려 원심 분리기. </li><li> 세포 펠렛을 방해하지 않도록 보장 라벨 매체를 대기음. </li><li> PBS로 세포를 씻으십시오. 세포를 피펫 아래로, 세포 펠렛을 휴식해야하고. </li><li> 후자의 두 단계 두 번 더 반복하므로 3 세척 단계 총 있습니다. </li><li> 세포를 카운트하고 세포 생존을 결정하기 위해 Trypan 파랑 테스트를 수행합니다. 이것은 우리가 여기서 설명하지 않을 것을 표준 세포 배양 프로토콜입니다. </li><li> 당신의 세포는 현재 광학 영상기에 몇 군데 준비하고 있습니다. </li></ol> 형광 염료의 ICG와 인간 배아 줄기 세포의 라벨링 <ol><li> 인간 배아 줄기 세포를 라벨에 대한 절차를 시작하려면 대비 에이전트가 그린 Indocyanine 준비합니다. transfection 에이전트 프로타민와 함께 섞는다. </li><li> Indocyanine 그린 파우더 1mg을 측정합니다. 디메틸 Sulfoxide (DMSO) 100 μL에 ICG 분말을 디졸브. </li><li> 혼합물에 Dulbecco의 수정 이글스 중간 (DMEM + 10% 소태아 세럼) 미디어 400 μL를 추가하고 잘 흔들어. 의 2mg/ml의 최종 농도이 결과는 그린 Indocyanine. </li><li> transfection 에이전트 프로타민를 추가합니다. 대비 에이전트 셔틀로 프로타민 행위, 그것이보다 효율적으로 세포에 도착 있도록. </li><li> 300 μL ICG 300 μL 혈청 무료 Dulbecco의 수정 이글스 매체, 10mg/ml의 농도에서 제공하는 5 μL 프로타민 황산을 섞는다. </li><li> 부드럽게 양식으로 복잡한 수 있도록 5 분 동안 새로운 transfection 솔루션을 흔들. </li><li> 라벨 솔루션은 준비가 된 것입니다. </li><li> hESC 10mm 페트리 접시에서 기존의 미디어를 대기음. </li><li> 사전 예열 혈청없는 DMEM의 5ml를 추가합니다. </li><li> 세포에 이전 준비 프로타민 / ICG 솔루션을 추가합니다. 37 인큐베이터에있는 접시를 배치하여 1 시간 보육을 시작 ° C. </li><li> 배양이 완료되면, 인큐베이터에서 접시를 제거하고 라벨 솔루션을 대기음. </li><li> 5 ML PBS로 접시를 rinsing하여 세포를 씻으십시오. </li><li> PBS를 기음과 0.25 %의 트립신의 5ml로 바꿉니다. ° C 5 분 trypsinization가 발생할 수 있도록 37 접시를 품어. 그것은 한동안 조금 매번 접시를 흔들 수 있도록 도와줍니다. </li><li> 부드럽게 남아있는 식민지를 깨고, 아래로 피펫합니다. </li><li> 접시에 KSR의 동일한 금액을 추가하여 트립신을 중화. </li><li> 15ml 튜브에 세포 솔루션을 전송 5 분 400 RCF에서 솔루션을 원심 분리기. </li><li> 전체 미디어 세포를 Resuspend. </li><li> 지금이 시점에서 대단히 짧은 시간이있다면, trypsinize 다시 원심 분리기. </li><li> 덩어리가없는 세포 솔루션 달성되면, 기존의 미디어를 기음과 미리 예열 전체 ESC 미디어의 10ml의 펠렛을 resuspend. </li><li> 이 시점에서, 그것은 hESCs에서 마우스 피더 세포를 분리하는 것이 필요합니다. 이것은 그것을 보장하기 위해 수행, 나중에, 오직 영상 줄기 세포가 발생합니다. </li><li> 이를 위해 일을 전송할젤라틴 코팅 10cm 접시에 전자 전지 솔루션입니다. </li><li> 37 ° C 배양기에 접시를 넣어 45 분 동안 앉아 보자. 이 시간 동안 접시를 방해하지 않도록하십시오. 자, 피더는 음식을 준수하고 줄기 세포는하지 않습니다. </li><li> 페트리 접시 밖으로 솔루션을 전송합니다. 지금 우리는 인간의 배아 줄기 세포의 표시 단일 세포의 해결책을 가지고 있습니다. </li><li> 세포는 현재 계산 수 있으며 Trypan 파랑 시험은 그들을 수행할 수 있습니다. </li><li> 세포가 몇 군데 준비 있습니다! </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>

오이는 형광의 감지에 따라 비교적 새로운 이미징 기술입니다. 오이는 radiotracer 기반 이미징 기술을 같이 구분하지만, 모든 방사선 노출과 관련되지 않았습니다. 오이는이를 대상 사이트에 셀 마이 그 레이션에 대한 통찰력을 제공하는 비 invasively하고 반복적으로 추적 세포의 효과적인 수단을 제공합니다. 기술 중 하나는 주요 제한은 생체내에 형광 프로브의 제한된 조직 침투입니다. 따라서 피부 표면 5-10cm에 대한보다 깊은 조직의 추적 축적은, 감지되지 않을 수도 있습니다. 현재 임상 응용 프로그램 등, 내시경 심혈관 및 retinae 이미징 (Funovics 외. 2003)로 표면 기술로 제한되어 있습니다하지만, 연구의 도메인 생체내 세포 추적에 의해 제공하는 거대한 가능성의 인식을 통해 확장하는 것입니다.

<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

광학 이미징 (오이)는 새로운 줄기 세포 기반 요법의 생체내 모니터링을 위해 쉽고, 빠르고 저렴한 도구입니다. 이 기술은 형광 염료, 특정 대상 기관 또는 pathologies들의 축적의 라벨 세포 및 시각화의 후속 정맥 주사와 함께 줄기 세포의 생체내 전 라벨에 따라 달라집니다. 제공 기술나요 (C67H103CIN2O3S)와 우리가 인간 배아의 줄기 세포를 (hESC) 라벨을 어떻게 FDA는 형광 염료가 그린 (ICG)을 Indocyanine 승인을 우리가 lipophilic 형광 염료와 함께 간단하게 부화하여 인간 mesenchymal 줄기 세포 (hMSC)를 분류하는 방법을 보여줍니다. 이해 메커니즘은 인지질의 세포 막 이중층에 걸쳐 lypophilic 염료의 준수 및 확산을 통해이다. 세포와 같은 혈청 함유 미디어 부화 반대로 혈청없는 미디어의 염료와 incubated하는 경우 라벨의 효율은 일반적으로 향상됩니다. 또한, transfection 에이전트 프로타민 황산의 추가가 대비 에이전트 이해를 크게 향상시킵니다.

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

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

이 동영상은 형광 염료와 인간 배아 줄기 세포 및 mesenchymal 줄기 세포의 라벨에 대한 기술을 보여줍니다. 이 기술은 광학 이미징과 이식 줄기 세포의 생체내 추적에와 형광 현미경과 histopathological 상관 관계에 대해 사용할 수 있습니다.

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 조회수.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. 이 동영상은 형광 염료와 인간 배아 줄기 세포 및 mesenchymal 줄기 세포의 라벨에 대한 기술을 보여줍니다. 이 기술은 광학 이미징과 이식 줄기 세포의 생체내 추적에와 형광 현미경과 histopathological 상관 관계에 대해 사용할 수 있습니다.

비디오: 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.

멀티 컬러 형광 기자 마우스를 사용하여 각막 세포의 리니지 추적하는 방법

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

연구

발생학

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.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

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.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

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

생물학

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.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

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.

3차원 스페로이드 배양에서 줄기와 같은 세포의 분리

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

암 연구학

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.

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

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

신경과학

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.

Vivo에서 인간 지방 유래 중간 엽 줄기 세포 형광 질 성 막 염료와 쥐 무릎 관절염 모델에서의 추적

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.

유방 종양 미세환경의 생체 내 이미징을 위한 라벨 없는 세분화 접근법

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.

마우스 망막 순환에서 유동 역학을 연구하기위한 적혈구 및 백혈구의 형광 염료 표지

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

의학

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.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

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