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Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain.

ABSTRACT: Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.

SUBMITTER: Daadi MM

PROVIDER: S-EPMC2835224 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain.

Daadi Marcel M MM Li Zongjin Z Arac Ahmet A Grueter Brad A BA Sofilos Marc M Malenka Robert C RC Wu Joseph C JC Steinberg Gary K GK

Molecular therapy : the journal of the American Society of Gene Therapy 20090512 7

Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a ...[more]

PMID: 19436269

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Project description:Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 ?g/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI.The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials.

Project description:Quantitative magnetic resonance imaging (MRI) can serially and noninvasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also noninvasively monitor stem cell migration following iron oxide prelabeling. Reports have shown that neural stem cells (NSCs) may help mediate neuroprotection or stimulate neuroreparative responses in adult and neonatal models of ischemic injury. We investigated the ability of high-field MRI to monitor and noninvasively quantify the migration, proliferation, and location of iron oxide-labeled NSCs over very long time periods (58 weeks) in real time while contemporaneously correlating this activity with the evolving severity and extent of neural damage.Labeled clonal murine NSCs (mNSCs) were implanted 3 days after unilateral HII in 10-day-old rat pups into the contralateral striatum or ventricle. We developed methods for objectively quantifying key aspects of dynamic NSC behavior (eg, viability; extent, and speed of migration; degree of proliferation; extent of integration into host parenchyma). MRI images were validated with histological and immunohistochemical assessments.mNSCs rapidly migrated (100 ?m/day) to the lesion site. Chains of migrating NSCs were observed in the corpus callosum. In pups subjected to HII, though not in intact control animals, we observed a 273% increase in the MR-derived volume of mNSCs 4 weeks after implantation (correlating with the known proliferative behavior of endogenous and exogenous NSCs) that slowly declined over the 58-week time course, with no adverse consequences. Large numbers of now quiescent mNSCs remained at the site of injury, many retaining their iron oxide label.Our studies demonstrate that MRI can simultaneously monitor evolving neonatal cerebral injury as well as NSC migration and location. Most importantly, it can noninvasively monitor proliferation dynamically for prolonged time periods. To be able to pursue clinical trials in newborns using stem cell therapies it is axiomatic that safety be insured through the long-term real time monitoring of cell fate and activity, particularly with regard to observing unanticipated risks to the developing brain. This study supports the feasibility of reliably using MRI for this purpose.

Project description:BackgroundMagnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is (19)F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with (19)F as well as detected and quantified in vitro and after brain implantation.Methodology/principal findingsHuman NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with (19)F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the (19)F MR images.Conclusion/significanceOur results show that NSCs can be efficiently labeled with (19)F with little effects on viability or proliferation and differentiation capacity. We show for the first time that (19)F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.

Dataset Information

Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain.

Publications

Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain.

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