Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering.

For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy.

Microglia are phagocytic cells that are chemoattracted by brain tumors and can represent up to 70% of the tumor cell population. To get insight into gene therapy against glioma, we decided to take advantage of those microglia properties and to use those cells as vehicles to transport simultaneously a suicide gene (under the control of a heat-sensitive promoter) and contrast agents to localize them by magnetic resonance imaging before applying any therapeutic treatment. Thymidine kinase (TK) expression and its functionality after gancyclovir administration were investigated. After the heat shock (44 degrees C and 20 min), TK was expressed in 50% of the cells. However, after gancyclovir treatment, 90% of the cells died by apoptosis, showing an important bystander effect. Then, the cells were incubated with new lanthanide contrast agents to check both their potential toxicity and their MR properties. Results indicate that the nanoparticles did not induce any cell toxicity and yield a hypersignal on MR images at 4.7 T. These in vitro experiments indicate that microglia are good candidates as vectors in gene therapy against brain tumors. Finally, microglia containing gadolinium-grafted nanoparticles were injected in the close vicinity of C6 tumor, in a mouse. The hyperintensive signal obtained on in vivo images as well as its retention time show the potential of the novel contrast agents for cellular imaging.

Lactate involvement in neuron-glia metabolic interaction: (13)C-NMR spectroscopy contribution.

Glucose is commonly admitted to be the main substrate for brain energy requirement. However, it has been recently proposed that lactate, generated from glucose via glycolysis, would be the oxidative substrate for neurons, particularly during neuronal activation, according to a mechanism called the astrocyte-neuron lactate shuttle hypothesis (ANLSH). In that mechanism, glutamate released in the synaptic cleft during brain activation is taken up by astrocytes. This uptake, via the glutamate/Na(+) transporter, induces the entry of sodium, which is then excluded from the astrocytes via the Na(+)/K(+) ATPase. This exclusion consumes ATP, which stimulates glycolysis and thus lactate formation in astrocytes. This lactate is then transferred to neurons where it is utilized as oxidative substrate. This review tries to gather the recent evidences that support this hypothesis and presents the contribution of NMR to this matter.

Effect of exogenous lactate on rat glioma metabolism.

Glioma-bearing rats were infused intravenously with a solution containing either [3-(13)C]lactate or both glucose and [3-(13)C]lactate for 20 min or 1 hr. Perchloric acid extracts of healthy and tumoral brain tissues were prepared and analyzed by (13)C- and (1)H-observed (13)C-edited nuclear magnetic resonance (NMR) spectroscopy to determine (13)C-label incorporation into brain tissue and glioma metabolites. Moreover, (13)C enrichments in blood lactate and glucose were determined from (1)H-NMR spectra. In the nontumoral tissue, (13)C labeling of amino acids indicated that [3-(13)C]lactate entered the brain and was metabolized. There was no labeling difference between the contralateral and the ipsilateral hemispheres. Lactate metabolism appeared more specifically neuronal, in agreement with our previous results obtained with normal rat brain (Bouzier et al. [2000] J. Neurochem. 75:480-486). In the glioma tissue, comparison of Ala C3, Glu C4, and Gln C4 labeling indicated that the contributions of blood glutamine and tricarboxylic acid (TCA) cycle to glutamate labeling were about 80% and 20%, respectively, after 1 hr of [3-(13)C]lactate infusion. In contrast, these contributions were about 10% and 90%, respectively, when [1-(13)C]glucose was infused in the absence of lactate. This indicated a major effect of the exogenous lactate on glioma metabolism, which may be due to the following process: The high blood lactate level might hinder the drain of glycolytic lactate produced inside the glioma and thus generate a change in redox potential such that the tumor cells are unable to restore it with oxidative phosphorylation. Thereafter, the high NADH level might inhibit glycolysis and the TCA cycle, and glutamine could become the major carbon source for glutamate labeling.