Fangjing Wang

Imaging of Mesenchymal Stem Cell Transplant by Bioluminescence and PET

Dynamic measurements of infused stem cells generally require animal euthanasia for single-time-point determinations of engraftment. In this study, we used a triple-fusion reporter system for multimodal imaging to monitor human mesenchymal stem cell (hMSC) transplants. Methods: hMSCs were transduced with a triple-fusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral vector. Transduced cells were assayed in vitro for the expression of each functional component of the triple-fusion reporter. Transduced and control hMSCs were compared for their potential to differentiate into bone, cartilage, and fat. hMSCs expressing the reporter were then loaded into porous, fibronectin-coated ceramic cubes and subcutaneously implanted into NOD-SCID mice along with cubes that were loaded with wild-type hMSCs and empty cubes. Mice were imaged repeatedly over 3 mo by bioluminescence imaging (BLI), and selected animals underwent CT and PET imaging. Results: Osteogenic, adipogenic, and chondrogenic potential assays revealed retained differentiation potentials between transduced and wild-type hMSCs. Signals from the cubes loaded with reporter-transduced hMSCs were visible by BLI over 3 mo. There was no signal from the empty or wild-type hMSC–loaded control cubes. PET data provided confirmation of the quantitative estimation of the number of cells at one spot (cube). Cubes were removed from some animals, and histologic evaluations showed bone formation in cubes loaded with either reporter-transduced or wild-type hMSCs, whereas empty controls were negative for bone formation. Conclusion: The triple-fusion reporter approach resulted in a reliable method of labeling stem cells for investigation in small-animal models by use of both BLI and small-animal PET imaging. It has the potential for translation into future human studies with clinical PET.

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

Human Mesenchymal Stromal Cells Attenuate Graft‐Versus‐Host Disease and Maintain Graft‐Versus‐Leukemia Activity Following Experimental Allogeneic Bone Marrow Transplantation

We sought to define the effects and underlying mechanisms of human, marrow‐derived mesenchymal stromal cells (hMSCs) on graft‐versus‐host disease (GvHD) and graft‐versus‐leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T‐cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC‐treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T‐cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T‐cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T‐cell proliferation, reduced TNFα, IFNγ, and IL‐10 but increased PGE2 levels. Indomethacin and E‐prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC‐mediated in vitro T‐cell suppression, confirming the role for PGE2. Furthermore, cyclo‐oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T‐cell proliferation likely through PGE2 induction. Stem Cells 2015;33:601–614

[(Methyl)1-11C]-Acetate Metabolism in Hepatocellular Carcinoma

PurposeStudies have established the value of [(methyl)1-11C]-acetate ([11C]Act) combined with 2-deoxy-2[18F]fluoro-d-glucose (FDG) for detecting hepatocellular carcinoma (HCC) using positron emission tomography (PET). In this study, the metabolic fate of [11C]Act in HCC was characterized.MethodsExperiments with acetic acid [1-14C] sodium salt ([14C]Act) were carried out on WCH-17 cells and freshly derived rat hepatocytes. PET scans with [11C]Act were also carried out on woodchucks with HCC before injection of [14C]Act. The radioactivity levels in different metabolites were quantified with thin-layer chromatography.ResultsIn WCH-17 cells, the predominant metabolite was phosphatidylcholine (PC). Regions of HCCs with the highest [11C]Act uptake had higher radioactivity accumulation in lipid-soluble compounds than surrounding hepatic tissues. In those regions, PC and triacylglycerol (TG) accumulated more radioactivity than in surrounding hepatic tissues.ConclusionsHigh [11C]Act uptake in HCC is associated with increased de novo lipogenesis. PC and TG are the main metabolites into which the radioactive label from [11C]Act is incorporated in HCC.

Imaging Lipid Synthesis in Hepatocellular Carcinoma with [Methyl-11C]Choline: Correlation with In Vivo Metabolic Studies

PET with [methyl-11C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG–avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Methods: Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-14C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio–high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. Results: PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89 ± 0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. Conclusion: HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5′-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

Transport and metabolism of radiolabeled choline in hepatocellular carcinoma.

Altered choline (Cho) metabolism in cancerous cells can be used as a basis for molecular imaging with PET using radiolabeled Cho. In this study, the metabolism of tracer Cho was investigated in a woodchuck hepatocellular carcinoma (HCC) cell line (WCH17) and in freshly derived rat hepatocytes. The transporter responsible for [(11)C]-Cho uptake in HCC was also characterized in WCH17 cells. The study helped to define the specific mechanisms responsible for radio-Cho uptake seen on the PET images of primary liver cancer such as HCC. Cells were pulsed with [(14)C]-Cho for 5 min and chased for varying durations in cold media to simulate the rapid circulation and clearance of [(11)C]-Cho. Radioactive metabolites were extracted and analyzed by radio-HPLC and radio-TLC. The Cho transporter (ChoT) was characterized in WCH17 cells. WCH17 cells showed higher (14)C uptake than rat primary hepatocytes. [(14)C]-Phosphocholine (PC) was the major metabolite in WCH17. In contrast, the intracellular Cho in primary hepatocytes was found to be oxidized to betaine (partially released into media) and, to a lesser degree, phosphorylated to PC. [(14)C]-Cho uptake by WCH17 cells was found to have both facilitative transport and nonfacilitative diffusion components. The facilitative transport was characterized by Na(+) dependence and low affinity (K(m) = 28.59 ± 6.75 μM) with partial energy dependence. In contrast, ChoT in primary hepatocytes is Na(+) independent and low affinity. Our data suggest that transport and phosphorylation of Cho are responsible for the tracer accumulation during [(11)C]-Cho PET imaging of HCC. WCH17 cells incorporate [(14)C]-Cho preferentially into PC. Conversion of [(14)C]-PC into phosphatidylcholine occurred slowly in vitro. Basal oxidation and phosphorylation activities in surrounding hepatic tissue contribute to the background seen in [(11)C]-Cho PET images.

Cross‐species hybridization of woodchuck hepatitis viral infection‐induced woodchuck hepatocellular carcinoma using human, rat and mouse oligonucleotide microarrays

Background and Aim:  We aimed to evaluate the transcriptional characteristics of viral infection‐induced woodchuck hepatocellular carcinoma (HCC), to compare the use of human, rat and mouse gene arrays for cross‐species hybridization, and to look into gene expression profiles in woodchuck HCC by the combined use of these arrays.