Questo progetto è stato sostenuto da una borsa SEED J. Leon Thal del California Institute per la Medicina Rigenerativa. Tobias Henning è stato finanziato da una borsa di studio di ricerca dal tedesco Research Association (DFG, SE 4578/1-2). Vogliamo riconoscere con gratitudine Meneses Juanito per i suoi consigli sulla cultura di cellule staminali embrionali umane.

L&#39;etichettatura di hMSC con Ferucarbotran Questa è la tecnica per l&#39;etichettatura di cellule staminali mesenchimali con Ferucarbotran, un ossido di ferro agente di contrasto per RM: <ol><li> Per iniziare, piastra delle cellule 18-24 ore prima della procedura di etichettatura T75 fiaschi ad una confluenza del 80%. Se si utilizza un piatto diversa cultura, questa è pari a circa 10000 cellule per cm 2. </li><li> Il giorno dopo, iniziare con la preparazione dei mezzi di etichettatura con l&#39;aggiunta di 30 ml di Resovist a 8 ml di siero libertà dei media. Questo etichetta uno T75 pallone in 80% confluenza, corrispondente a una concentrazione di 100 mg Fe / ml media. </li><li> Togliere il mezzo di coltura e lavare le cellule una volta con PBS o senza siero dei media. Questo viene fatto per sbarazzarsi di residui di proteine ​​del siero ed altri costituenti dei media che possono legare il mezzo di contrasto e di etichettatura di efficienza influenza. </li><li> Aggiungi ai media di etichettatura per il pallone e rimettere il pallone in incubatrice. </li><li> Dopo 2 ore, aggiungere 2 ml di FCS modo che la concentrazione finale del 20% FCS è raggiunto. Questo viene fatto per garantire che le cellule non ricevono lo stimolo a differenziare, ma invece crescere nel loro ambiente familiare. </li><li> Incubare le cellule oltre 18 ore. Questo è fatto meglio durante la notte. </li><li> Il giorno dopo, lavare le cellule con PBS, poi trypsinize come al solito. </li><li> Una volta trypsinized, la sospensione cellulare ha bisogno di essere lavati 3 volte per sbarazzarsi di residui, senza mezzo di contrasto nei media. Questo viene fatto con PBS centrifugando le cellule a 400 rcf per 5 minuti. </li><li> La finale pellet cellulare può essere risospeso ed è pronto per ulteriori esperimenti. </li><li> Contare le celle ora, come qualcuno potrebbe essere stato perso durante i passaggi precedenti lavaggio. Inoltre, eseguire test di vitalità a questo punto, utilizzando il test Trypan esclusione blu e prendere alcuni campioni per analisi di spettrometria per misurare l&#39;efficienza di etichettatura. </li></ol> Etichettatura delle cellule staminali embrionali umane con Ferumoxides <ol><li> Per iniziare la targhetta del hESC nei piatti 10cm, come al solito. Questi piatti sono pre-rivestiti con gelatina e sono cellule alimentatore irradiato su di loro. È possibile utilizzare questo protocollo per l&#39;alimentazione priva di culture pure. </li><li> Lasciate che i hESC attaccare e crescere per circa 3-4 giorni in modo da formare colonie di medie dimensioni. Durante questo tempo, in modo che le colonie non crescono alla grande, come le colonie più grandi sono più difficili da rompere più tardi. </li><li> Cellule differenziate possono contaminare queste culture, per cui a seconda della pulizia delle colonie, potrebbe essere necessario sbarazzarsi di culture differenziate da dissezione. </li><li> Preparare il supporto di etichettatura che consiste Ferumoxides ad una concentrazione di mezzi hESC 100μg/ml e pieno. Per questo, abbiamo mix 89μl Ferumoxides e 10 ml di media piena crescita. </li><li> Lavare il piatto una volta con i media completa. </li><li> Aggiungere 10 ml di etichettatura dei media per ogni piatto e incubare le cellule per 4 ore. </li><li> Ora, l&#39;etichettatura è completa. Nel passo successivo, è necessario lavare le cellule e ottenere una sospensione singola cella per un ulteriore uso. Per questo, prima lavare il piatto con PBS. </li><li> Poi, sostituire la PBS con 5 ml di tripsina 0,25% e incubare nell&#39;incubatore per 5 minuti, o fino a quando le cellule staccare. Aiuta a toccare il piatto di frequente. </li><li> Le cellule sono ormai staccati. Tenete presente che se il piatto conteneva le colonie più grandi ci potrebbero essere alcuni cespugli di sinistra. </li><li> Per sbarazzarsi di questi grumi, si può miscelare la sospensione intero una volta con una pipetta sierologica, poi lasciate riposare per altri 2 minuti. </li><li> Noi non usare una pipetta Eppendorf, succhiando come ripetuto delle cellule attraverso la punta sottile può rompere le membrane cellulari. </li><li> Se si osservano residui sono presenti, si può eseguire la sospensione cellulare attraverso un colino cellula 40μm. </li><li> Se tutto ha funzionato correttamente, una sospensione singola cellula esiste, ora che è mescolato con un sacco di detriti, provenienti dal rivestimento in gelatina, il CES-matrice e cellule morte. Per sbarazzarsi di questo, lavare le cellule due volte facendo girare giù a 400 rcf per 5 minuti e risospendere in mezzi di comunicazione completo. </li><li> Ora, il Comitato deve essere isolato dalle cellule alimentatore irradiati. Questo può essere fatto in modo efficiente dal placcatura la sospensione cellulare su un piatto rivestito di gelatina. </li><li> Se il piatto non è manipolato, tutte le cellule si sedimenta giù, ma gli alimentatori che attribuirà più veloce del CES. </li><li> Quindi, se il surnatante viene tolto dopo 45 minuti, dovrebbe contenere principalmente ESC, mentre gli alimentatori dovrebbe principalmente essere allegata al piatto. </li><li> In questo modo, una sospensione singola cella di magneticamente etichettato cellule staminali embrionali umane che possono essere utilizzati per ulteriori esperimenti si ottiene. </li><li> Come nel precedente protocollo, a questo punto, è necessario contare le cellule e prelevare campioni per valutazione della vitalità economica e la misurazione di efficienza etichettatura. </li></ol>

<ol>
	<li>Daldrup-Link, H. E., Rudelius, M., Oostendorp, R. A. J., Settles, M., Piontek, G., Metz, S., Heinzmann, U., Rummeny, E. J., Schlegel, J., Link, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Hematopoietic+Progenitor+Cells+with+MR+Contrast+Agents.">Targeting of Hematopoietic Progenitor Cells with MR Contrast Agents.</a> 228, 760-767 (2003).</li><li>Daldrup-Link, H. E., Rudelius, M., Piontek, G., Metz, S., Brauer, R., Debus, G., Corot, C., Schlegel, J., Link, T. M., Peschel, C., 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=Migration+of+iron+oxide+labeled+human+hematopoietic+progenitor+cells+in+a+xenotransplant+model:+in+vivo+monitoring+using+clinical+magnetic+resonance+imaging+equipment.">Migration of iron oxide labeled human hematopoietic progenitor cells in a xenotransplant model: in vivo monitoring using clinical magnetic resonance imaging equipment.</a> Radiology. 234, 197-205 (2005).</li><li>Henning, T. D., Saborowski, O., Golovko, D., Boddington, S., Bauer, J. S., Fu, Y., Meier, R., Pietsch, H., Sennino, B., McDonald, D. M., 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+labeling+with+the+positive+MR+contrast+agent.">Cell labeling with the positive MR contrast agent.</a> Gadofluorine M. Eur Radiol. 17, 1226-1234 (2007).</li></ol>

Un non-invasiva rappresentazione delle cellule staminali è fondamentale per il monitoraggio praticamente qualsiasi terapia a base di cellule staminali. Una migliore comprensione della cinetica in vivo delle cellule staminali etichettati, come visualizzato nelle immagini MR, potrebbe aprire la strada ad un razionale e un uso più efficace delle cellule staminali a base di terapie. Dal momento che usiamo mezzi di contrasto clinicamente applicabile e tecniche di imaging, i nostri protocolli presentati devono essere facilmente traducibili in a...

<table><tbody><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 Gelatin in PBS to final concentration of 0.1 %</td></tr><tr><td>PBS (Ca++ Mg++ freed)</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>FBS 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>Tool</td><td>BD Biosciences</td><td>352340</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</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>Ferumoxides (Endorem)</td><td>Reagent</td><td>Guerbet</td><td></td><td></td></tr><tr><td>Ferucarbotran (Resovist&amp;#174;)</td><td>Reagent</td><td>Bayer AG</td><td></td><td></td></tr></tbody></table>

labeling hescs and hmscs with iron oxide nanoparticles for non-invasive in vivo tracking with mr imaging

Negli ultimi anni la ricerca sulle cellule staminali ha portato ad una migliore comprensione della biologia dello sviluppo, malattie varie e il suo potenziale impatto sulla medicina rigenerativa. Un metodo non invasivo per monitorare le cellule staminali trapiantate in vivo avrebbe ripetutamente di migliorare notevolmente la nostra capacità di comprendere i meccanismi che la morte di controllo delle cellule staminali e di identificare fattori trofici e di vie di segnalazione che consentono di migliorare l&#39;attecchimento delle cellule staminali. RM ha dimostrato di essere uno strumento efficace per la rappresentazione in vivo di cellule staminali con vicino risoluzione anatomica microscopica. Al fine di individuare le cellule staminali con MR, le cellule devono essere etichettati con cellulari specifici mezzi di contrasto MR. A tal fine, le nanoparticelle di ossido di ferro, come le particelle di ossido di ferro superparamagnetiche (SPIO), vengono applicate, a causa della loro elevata sensibilità per il rilevamento delle cellule e la loro eccellente biocompatibilità. SPIO particelle sono composte da un nucleo di ossido di ferro e un cappotto destrano, carboxydextran o amido, e la funzione creando disomogeneità campo locale, che causano una diminuzione del segnale sulle immagini T2 MR. Questa presentazione dimostrerà le tecniche per l&#39;etichettatura di cellule staminali clinicamente applicabile con mezzi di contrasto RM per la successiva non invasive nel monitoraggio in vivo delle cellule etichettate con la RM.

Watch this Scientific Journal Video about Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging at JoVE.com

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

Per la valutazione delle terapie con cellule staminali nuovo, è importante non invasiva traccia le cellule iniettate in vivo. Questo video vi mostrerà come etichetta cellule staminali mesenchimali ed embrionali con il ferro mezzi di contrasto a base di ossidi in vivo per la successiva RM in vivo.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

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

labeling-hescs-hmscs-with-iron-oxide-nanoparticles-for-non-invasive

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.8K Views.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. Per la valutazione delle terapie con cellule staminali nuovo, è importante non invasiva traccia le cellule iniettate in vivo. Questo video vi mostrerà come etichetta cellule staminali mesenchimali ed embrionali con il ferro mezzi di contrasto a base di ossidi in vivo per la successiva RM in vivo.

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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Bioimmagini registrato di Nanomateriali per il monitoraggio diagnostico e terapeutico

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Ricerca

Bioingegneria

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Etichettatura cellule staminali con Ferumoxytol, un approvati dalla FDA nanoparticelle di ossido di ferro

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Medicina

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

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 of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con Optical Imaging

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 ...

Biologia

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoraggio migrazione delle cellule dendritiche usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Immunologia e infezioni

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.

Imaging multimodale impianto di cellule staminali nel sistema nervoso centrale di topi

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

Neuroscienze

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Etichettatura delle cellule e Targeting con superparamagnetico ossido di ferro nanoparticelle

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

L&#39;impianto di Ferumoxides etichetta cellule staminali mesenchimali in difetti della cartilagine

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Monitoraggio delle cellule staminali mesenchymale con etichetta chimica dell'ossido di ferro superparamagnetico utilizzando la risonanza magnetica dopo la consegna intranasale in un modello murino traumatico da lesioni cerebrali

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

Riepilogo

Esplora Altri Video

Capitoli in questo video