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, ...

University of Antwerp

Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice.

La transplantation de cellules est susceptible de devenir un important outil th&eacute;rapeutique pour le traitement de diverses blessures traumatiques et isch&eacute;miques au syst&egrave;me nerveux central (CNS). Toutefois, dans de nombreuses &eacute;tudes de th&eacute;rapie cellulaire pr&eacute;cliniques, imagerie assist&eacute;e par g&egrave;ne de journaliste d&#39;implants cellulaires dans le CNS et des potentiels g&egrave;ne rapporteur et/ou cell-based immunog&eacute;nicit&eacute;, restent encore des sujets de recherche difficile.

Journaliste des cellules stromales de moelle osseuse exprimant le g&egrave;ne sont immunitaire-tol&eacute;r&eacute; apr&egrave;s l&#39;implantation du syst&egrave;me nerveux central de souris immunocomp&eacute;tentes syng&eacute;niques.

Trapianto di cellule &egrave; destinato a diventare un importante strumento terapeutico per il trattamento di varie lesioni traumatiche e ischemici al sistema nervoso centrale (SNC). Tuttavia, in molti studi di terapia cellula pre-clinici, reporter gene-assistita imaging cellulari impianti nel CNS e potenziale gene reporter e/o cell-based immunogenicit&agrave;, rimangono ancora difficili temi di ricerca.

Reporter gene-esprimendo derivate da midollo osseo cellule stromali sono tollerati immunitario seguente impianto nel sistema nervoso centrale dei topi immunitarie Carlo.

While neural stem cells (NSCs) are widely expected to become a therapeutic agent for treatment of severe injuries to the central nervous system (CNS), currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether IV administration of allogeneic NSC can improve experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human multiple sclerosis (MS). For this, we cultured adherently growing luciferase-expressing NSCs (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labeling with green fluorescent micron-sized iron oxide particles (f-MPIO-labeled NSC-Luc) or lentiviral transduction with the enhanced green fluorescent protein (eGFP) reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analyzed by real-time bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP at day 7 postinduction with or without daily immunosuppressive therapy (cyclosporine A, CsA). During a follow-up period of 20 days, the observed clinical benefit could be attributed solely to CsA treatment. In addition, histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuroinflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV-administered NSC-Luc/eGFP revealed cell retention in lung capillaries as soon as 1-min postinjection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSCs currently has limited or no therapeutic potential for neuroinflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSCs have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and-in contrast to several other reports-will most likely be highly complex.

Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice?

Alors que les cellules souches neurales (CNS) devraient largement pour devenir un agent th&eacute;rapeutique pour le traitement de blessures graves pour le syst&egrave;me nerveux central (CNS), actuellement il y a seulement quelques d&eacute;taill&eacute;es &eacute;tudes pr&eacute;cliniques liant le sort de la cellule avec les r&eacute;sultats exp&eacute;rimentaux. Dans cette &eacute;tude, nous avons cherch&eacute; &agrave; v&eacute;rifier si l&#39;administration IV de NSC allog&eacute;nique peut am&eacute;liorer l&#39;enc&eacute;phalomy&eacute;lite allergique exp&eacute;rimentale (EAE), un mod&egrave;le animal bien &eacute;tabli pour la scl&eacute;rose en plaques (MS). Pour ce faire, nous avons cultiv&eacute; adherently de plus en plus les CNS (NSC-Luc), qui affiche une morphologie uniforme et le profil d&#39;expression de la membrane et marqueurs intracellulaires et qui affiche une potentielle de diff&eacute;renciation in vitro dans les neurones et les astrocytes exprimant lucif&eacute;rase. &Eacute;tiquetage suivant avec vert oxyde de fer taille micron fluorescent particules (f-MPIO-&eacute;tiquet&eacute; NSC-Luc) ou des g&egrave;nes transduction avec le g&egrave;ne rapporteur de la prot&eacute;ine fluorescente verte am&eacute;lior&eacute;e (eGFP) (NSC-Luc/eGFP), implantation de cellule exp&eacute;riences ont d&eacute;montr&eacute; la capacit&eacute; intrins&egrave;que de la survie du NSC adherently cultiv&eacute; dans le syst&egrave;me nerveux central de souris syng&eacute;niques, telle qu&#39;analys&eacute;e par imagerie en temps r&eacute;el de bioluminescence (BLI), imagerie par r&eacute;sonance magn&eacute;tique (IRM) et une analyse histologique. Ensuite, EAE a induit chez les souris C57BL/6, suivies de l&#39;administration IV de NSC-Luc/eGFP &agrave; postinduction jour 7 avec ou sans traitement immunosuppresseur quotidiens (cyclosporine A, CsA). Pendant une p&eacute;riode de 20 jours de suivi, les avantages cliniques observ&eacute;es peuvent &ecirc;tre attribu&eacute;es uniquement au traitement de la CsA. En outre, l&#39;analyse histologique a montr&eacute; l&#39;absence de NSC-Luc/eGFP dans les sites de la neuroinflammation. Afin d&#39;enqu&ecirc;ter sur l&#39;absence de th&eacute;rapeutique potentiel, analyse de biodistribution BLI de IV-administr&eacute; NSC-Luc/eGFP a r&eacute;v&eacute;l&eacute; r&eacute;tention cellulaire dans les capillaires pulmonaires comme bient&ocirc;t aussi 1-min apr&egrave;s l&#39;injection, ce qui entra&icirc;ne une inflammation massive et l&#39;apoptose dans les tissus pulmonaires. En r&eacute;sum&eacute;, nous concluons que l&#39;administration IV de CNS actuellement a limit&eacute; ou aucun potentiel th&eacute;rapeutique pour les maladies th&eacute;rapeutiques chez les souris et sans doute aussi pour MS humaine. Toutefois, compte tenu du fait que greff&eacute;s CNS ont une capacit&eacute; de survie intrins&egrave;que dans le SNC, leur exploitation th&eacute;rapeutique devrait &ecirc;tre davantage &eacute;tudi&eacute;e, et-contrairement &agrave; plusieurs autre rapports-sera tr&egrave;s probablement &ecirc;tre tr&egrave;s complexe.

Clinique potentiel de livraison par voie intraveineuse les cellules souches neurales pour le traitement de la maladie chez les souris neuroinflammatoire ?

Mentre le cellule staminali neurali (NSCs) sono ampiamente previsto di diventare un agente terapeutico per il trattamento di gravi lesioni al sistema nervoso centrale (SNC), attualmente ci sono solo alcuni dettagliati studi preclinici che collega il destino delle cellule con risultato sperimentale. In questo studio, abbiamo mirato a verificare se la somministrazione IV di NSC allogenico pu&ograve; migliorare l&#39;encefalomielite autoimmune sperimentale (EAE), un consolidato modello animale umano sclerosi multipla (MS). Per questo, abbiamo colto adherently crescente luciferase-esprimendo NSCs (NSC-Luc), che una morfologia uniforme e un profilo di espressione di membrana e intracellulari marcatori, mentre che visualizzata una differenziazione in vitro potenziale in neuroni e astrociti. Seguente etichettatura con particelle di verde fluorescente dimensioni micron ferro ossido (f-MPIO-etichettati NSC-Luc) o trasduzione lentivirale con il gene reporter avanzata proteina fluorescente verde (eGFP) (NSC-Luc/eGFP), l&#39;impianto delle cellule esperimenti hanno dimostrato la capacit&agrave; di sopravvivenza intrinseco del NSC adherently colta nel SNC di Carlo topi, come analizzato da imaging in tempo reale di bioluminescenza (BLI), imaging a risonanza magnetica (MRI) e l&#39;analisi istologica. Successivamente, EAE &egrave; stato indotto nei topi C57BL/6 seguiti da somministrazione IV di NSC-Luc/eGFP al postinduction giorno 7 con o senza terapia immunosoppressiva quotidiana (ciclosporina A, CsA). Durante un periodo di follow-up di 20 giorni, il beneficio clinico osservato potrebbe essere attribuito unicamente al trattamento di CsA. Inoltre, l&#39;analisi istologica ha dimostrato l&#39;assenza di Luc/NSC-eGFP presso siti di neuroinflammation. Al fine di indagare l&#39;assenza di terapeutica potenziale, analisi di biodistribuzione BLI di somministrato IV Luc/NSC-eGFP rivelato ritenzione delle cellule nei capillari polmonari come presto come 1-min postinjection, con conseguente infiammazione massiccia e apoptosi nel tessuto polmonare. In sintesi, possiamo concludere che la somministrazione IV di NSCs attualmente ha limitato o nessun potenziale terapeutico per la malattia di neuroinflammatory nei topi e presumibilmente anche per MS umana. Tuttavia, tenuto conto del fatto che innesta NSCs hanno una capacit&agrave; di sopravvivenza intrinseco nel SNC, loro sfruttamento terapeutico dovrebbe essere ulteriormente indagato, e in contrasto con diversi altri rapporti-sar&agrave; pi&ugrave; probabile essere altamente complessi.

Potenziale clinico della consegna per via endovenosa delle cellule staminali neurali per il trattamento della malattia neuroinflammatory nei topi?

Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.

Recognition of cellular implants by the brain's innate immune system.

Actuellement, beaucoup d&#39;attention est accord&eacute;e au d&eacute;veloppement de th&eacute;rapies cellulaires pour le traitement des l&eacute;sions du syst&egrave;me nerveux central (SNC). Diverses cellules implantation strat&eacute;gies, soit pour remplacer directement les tissus nerveux endommag&eacute;s ou pour cr&eacute;er un environnement neuroregenerative, sont propos&eacute;es pour r&eacute;tablir le fonctionnement du cerveau ayant une d&eacute;ficience. Toutefois, en raison de la complexit&eacute; de la CNS, il devient clair que la contribution de l&#39;implantation de cellules dans le cerveau s&#39;agira principalement d&#39;une mani&egrave;re favorable. En outre, compte tenu de la d&eacute;pendance temporelle du d&eacute;veloppement neural durant la vie embryonnaire et postnatale, cellulaire aux implants, soi ou non-soi, aura vraisemblablement d&#39;interagir pendant une p&eacute;riode prolong&eacute;e de temps avec les tissus nerveux sain et bless&eacute;s. Ce dernier implique aussi la reconnaissance potentielle des implants cellulaires par le syst&egrave;me immunitaire inn&eacute; du cerveau. Dans cette revue, nous mettront l&#39;accent sur les observations pr&eacute;cliniques chez les rongeurs, en ce qui concerne la reconnaissance et l&#39;immunog&eacute;nicit&eacute; d&#39;autologue, allog&eacute;niques et cellulaire x&eacute;nog&eacute;nique implants dans le SNC des h&ocirc;tes immunitaires-comp&eacute;tentes. Ensemble, nous sugg&eacute;rons ici qu&#39;une &eacute;tude approfondie de l&#39;interaction entre les greffes cellulaires et le syst&egrave;me immunitaire inn&eacute; du cerveau sera in&eacute;vitable avant la transplantation de cellules clinique dans le syst&egrave;me nerveux central peut &ecirc;tre effectu&eacute;e avec succ&egrave;s.

Reconnaissance des cellulaires des implants par syst&egrave;me immunitaire inn&eacute; du cerveau.

Attualmente, molta attenzione &egrave; data allo sviluppo di terapie cellulari per il trattamento delle lesioni del sistema nervoso centrale (SNC). Sono proposte diverse celle l&#39;impianto strategie, sostituire direttamente i tessuti neurali danneggiati o per creare un ambiente neuroregenerative, per ripristinare la funzione cerebrale compromessa. Tuttavia, a causa della complessit&agrave; del sistema nervoso centrale, essa sta diventando chiaro che il contributo dell&#39;impianto di cellule nel cervello principalmente agir&agrave; in maniera solidale. Inoltre, data la dipendenza dal tempo di sviluppo neurale durante la vita embrionale e postnatale, cellular impianti, self o non-s&eacute;, molto probabilmente dovr&agrave; interagire per un periodo prolungato di tempo con tessuto neurale sia sano e ferito. Quest&#39;ultimo implica anche potenziale riconoscimento delle protesi cellulare dal sistema immunitario innato del cervello. In questa recensione, ci metter&agrave; in risalto sulle osservazioni precliniche in roditori, per quanto riguarda il riconoscimento e l&#39;immunogenicit&agrave; del trapianto autologo, allogenico e xenogenici cellulare impianti nel SNC di host immune-competente. Presi insieme, consigliamo qui che sar&agrave; inevitabile un profondo studio dell&#39;interazione tra innesti cellulare e sistema immunitario innato del cervello prima che il trapianto di cellule clinico nel SNC pu&ograve; essere eseguito con successo.

Riconoscimento del cellulare gli impianti dal sistema immunitario innato del cervello.

Sviluppo di strategie di imaging multimodale attualmente &egrave; della massima importanza per la convalida di studi terapia preclinica della cellula formativa.

Etichettatura di Luciferase/eGFP-esprimendo derivate da midollo osseo cellule stromali con particelle fluorescenti ossido di ferro di dimensioni micron migliora imaging multimodale quantitativa e qualitativa della cellular innesti in vivo.

Development of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies.

Labeling of Luciferase/eGFP-expressing bone marrow-derived stromal cells with fluorescent micron-sized iron oxide particles improves quantitative and qualitative multimodal imaging of cellular grafts in vivo.

&Eacute;laboration de strat&eacute;gies d&#39;imagerie multimodales est actuellement de la plus haute importance pour la validation des &eacute;tudes de th&eacute;rapie pr&eacute;clinique des cellules souches.

Marquage de la lucif&eacute;rase/eGFP-exprimant les cellules stromales d&eacute;riv&eacute;es de la moelle osseuse avec des particules d&#39;oxyde de fer de taille micron fluorescent am&eacute;liore l&#39;imagerie multimodale quantitative et qualitative des greffes cellulaires in vivo.

Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brain's innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand-and eventually control-cell graft-induced tissue damage and activation of the brain's innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.

Cell type-associated differences in migration, survival, and immunogenicity following grafting in CNS tissue.

La transplantation de cellules a &eacute;t&eacute; sugg&eacute;r&eacute;e pour afficher plusieurs effets neuroprotecteurs et/ou neuroregenerative dans des mod&egrave;les animaux du traumatisme du syst&egrave;me nerveux central (SNC). Cependant, tandis que la plupart des &eacute;tudes indiquent sur les observations cliniques, actuellement on en sait peu sur le sort r&eacute;el des populations cellule greff&eacute;es et si ou comment le syst&egrave;me immunitaire inn&eacute; du cerveau, essentiellement r&eacute;alis&eacute; par la microglie activ&eacute;e et les astrocytes, interagit avec cellulaire autologue implants. Dans cette &eacute;tude, nous avons greff&eacute; bien caract&eacute;ris&eacute; les cellules souches neurales, fibroblastes embryonnaires de souris, cellules dendritiques, moelle osseuse de cellules mononucl&eacute;aires et populations de spl&eacute;nocytes, tous isol&eacute;s ou de cultures de C57BL/6-eGFP souris transg&eacute;niques, l&#39;externa capsula (CE) de chez la souris C57BL/6 et sous CE enflamm&eacute;e/demyelinated de souris C57BL/6 cuprizone-trait&eacute;es. Deux semaines postgrafting, une analyse histologique multicolore quantitative approfondie a &eacute;t&eacute; effectu&eacute;e dans l&#39;ordre (i) pour quantifier la toxicit&eacute;, la migration, survie et localisation de greffe de cellules et (ii) de caract&eacute;riser endog&egrave;nes CNS des r&eacute;ponses immunitaires contre les greffes de cellules diff&eacute;rentes. R&eacute;sultats obtenus indiquent la d&eacute;pendance sur le type de cellules greff&eacute;e: (i) par le degr&eacute; de la migration de greffon cellulaire, survie et la toxicit&eacute; et (ii) une organisation diff&eacute;rente de la r&eacute;ponse immunitaire endog&egrave;ne. Selon ces observations, nous garantissons que des recherches plus pouss&eacute;es devraient &ecirc;tre entrepris pour comprendre- et &eacute;ventuellement contr&ocirc;le-cellulaire induite par le greffon tissus et activation du syst&egrave;me immunitaire inn&eacute; du cerveau. Ce dernier sera in&eacute;vitable avant la greffe de cellules dans le syst&egrave;me nerveux central peut &ecirc;tre effectu&eacute;e en toute s&eacute;curit&eacute; et avec succ&egrave;s en milieu clinique.

Cellule type associ&eacute;e &agrave; des diff&eacute;rences dans la migration, la survie et immunog&eacute;nicit&eacute; apr&egrave;s greffage dans le tissu de la CNS.

Trapianto di cellule &egrave; stato suggerito per visualizzare diversi effetti neuroprotettivi e/o neuroregenerative in modelli animali di trauma del sistema nervoso centrale (SNC). Tuttavia, mentre la maggior parte dei studi riportano su osservazioni cliniche, attualmente poco &egrave; noto riguardo la sorte effettiva delle popolazioni delle cellule innestate e se o come sistema immunitario innato del cervello, principalmente diretto da attivato microglia e astrociti, interagisce con cellulari autologhi impianti. In questo studio, abbiamo innestato ben caratterizzate delle cellule staminali neurali, fibroblasti embrionali del mouse, cellule dendritiche, cellule mononucleate del midollo osseo e splenocyte popolazioni, tutti isolati o colti da C57BL/6-eGFP topi transgenici, sotto la capsula externa (CE) di topi C57BL/6 sani e sotto l&#39;infiammato/demyelinated CE di topi C57BL/6 cuprizone-trattati. Due settimane postgrafting, un&#39;analisi istologica multicolor quantitativa estesa &egrave; stata eseguita in ordine (i) per quantificare la tossicit&agrave;, migrazione, sopravvivenza e localizzazione dell&#39;innesto delle cellule e (ii) per caratterizzare endogene risposte immunitarie di CNS contro gli innesti di cellule differenti. Risultati ottenuti indicano la dipendenza del tipo di cellule innestate: (i) un diverso grado di migrazione innesto delle cellule, la sopravvivenza e tossicit&agrave; e (ii) un&#39;organizzazione diversa della risposta immunitaria endogeno. Sulla base di queste osservazioni, garantiamo che dovrebbe essere effettuate ulteriori ricerche per capire- e alla fine danno ai tessuti indotta da innesto controllo-cella e attivazione del sistema immunitario innato del cervello. Quest&#39;ultimo sar&agrave; inevitabile prima cella innesto nel SNC pu&ograve; essere eseguita in modo sicuro e con successo in situazioni cliniche.

Irene Bergwerf

Bio Imaging Lab

University of Antwerp

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

Irene Bergwerf

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Bio Imaging Lab

University of Antwerp

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

Bio Imaging Lab