Article

Use of Bioluminescent Imaging to Assay the Transplantation of Immortalized Human Fetal Hepatocytes Into Mice

Transplant Research Institute, UC Davis Medical Center, Sacramento, CA 95817, USA.
Cell Transplantation (Impact Factor: 3.57). 02/2008; 17(8):899-909. DOI: 10.3727/096368908786576471
Source: PubMed

ABSTRACT Noninvasive serial monitoring of the fate of transplanted cells would be invaluable to evaluate the potential therapeutic use of human hepatocyte transplantation. Therefore, we assessed the feasibility of bioluminescent imaging using double or triple fusion lentiviral vectors in a NOD-SCID mouse model transplanted with immortalized human fetal hepatocytes. Lentiviral vectors driven by the CMV promoter were constructed carrying reporter genes: firefly luciferase and green fluorescence protein with or without herpes simplex virus type 1 thymidine kinase. Human fetal hepatocytes immortalized by telomerase reconstitution (FH-hTERT) were successfully transduced with either of these fusion vectors. Two million stably transduced cells selected by fluorescence-activated cell sorting were injected into the spleens of NOD-SCID mice pretreated with methylcholanthrene and monocrotaline. The transplanted mice were serially imaged with a bioluminescence charged-coupled device camera after D-luciferin injection. Bioluminescence signal intensity was highest on day 3 (6.10 +/- 2.02 x 10(5) p/s/cm2/sr, mean +/- SEM), but decreased to 2.26 +/- 1.54 x 10(5) and 7.47 +/- 3.09 x 10(4) p/s/cm2/sr on day 7 and 10, respectively (p = 0.001). ELISA for human albumin in mice sera showed that levels were similar to those of control mice on day 2 (3.25 +/- 0.92 vs. 2.84 +/- 0.59 ng/ml, mean +/- SEM), peaked at 18.04 +/- 3.11 ng/ml on day 7, and decreased to 8.93 +/- 1.40 and 3.54 +/- 0.87 ng/ml on day 14 and 21, respectively (p = 0.02). Real-time quantitative RT-PCR showed gene expression levels of human albumin, alpha1-antitrypsin, and transferrin in mouse liver were 60.7 +/- 6.5%, 26.0 +/- 1.4%, and 156.8 +/- 62.4% of those of primary human adult hepatocytes, respectively, and immunohistochemistry revealed cells with human albumin and alpha1-antitrypsin expression in the mouse liver. In conclusion, our study demonstrated that bioluminescent imaging appears to be a sensitive, noninvasive modality for serial monitoring of transplanted hepatic stem cells.

0 Followers
 · 
98 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Absorption maxima of firefly oxyluciferin (Oxyln) were calculated by taking into account all atoms (10,467-10,473) of Luciola cruciata luciferase (Luc) containing Oxyln, a cofactor adenosine monophosphate (AMP) and water molecules based on its X-ray diffraction data (PDB code: 2D1R). Energy minimization and absorption peak calculations were executed by the Quantum/Molecular Mechanics (QM/MM) method, and by the Intermediated Neglect of Differential Overlap for Spectroscopy (INDO/S) method, respectively. The present study focused on the charge of AMP present in the catalytic center of Luc and investigated the effect of its change on the absorption maxima of Oxyln. As a result, the tendencies of the absorption peaks were found to differ greatly according to the protonation state of Oxyln and to depend on the AMP charge: the absorption maxima of the monoanion and the neutral Oxyln shifted to the longer-wavelength region (approximately 600 nm) and to the shorter-wavelength region (around 400-500 nm), respectively. Although this trend does not differ from the calculation results for the isolated Oxyln, directly taking into account the specific interaction with the catalytic center of Luc, the absorption maxima of the monoanion and the neutral Oxyln shifted to the longer-wavelength region. The absorption maxima of both monoanion and neutral Oxyln shifted toward the longer-wavelength region on reducing the AMP negative charge from dianion to neutral. According to the results obtained by our "Full-QM" calculations considering the Oxyln-Luc interaction, it was concluded that the red light (similar to 620 nm) and the yellow-green light (similar to 560 nm) emission for the acidic and the neutral/basic pH, respectively, could be assigned to the monoanion and the neutral Oxyln. It was also concluded that the charge of AMP significantly affects the luminescent color.
    Computational and Theoretical Chemistry 10/2014; 1045:93–98. DOI:10.1016/j.comptc.2014.07.001 · 1.37 Impact Factor
  • Source
    01/2010; 1(3):113-114. DOI:10.3727/215517910X557138
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Presently, orthotopic liver transplant is the major therapeutic option for patients affected by primary liver diseases. This procedure is characterized by major invasive surgery, scarcity of donor organs, high costs, and lifelong immunosuppressive treatment. Transplant of hepatic precursor cells represents an attractive alternative. These cells could be used either for allogeneic transplantation or for autologous transplant after ex vivo genetic modification. We used stromal cells isolated from adipose tissue (AT-SCs) as platforms for autologous cell-mediated gene therapy. AT-SCs were transduced with lentiviral vectors expressing firefly luciferase, allowing for transplanted cell tracking by bioluminescent imaging (BLI). As a complementary approach, we followed circulating human α1-antitrypsin (hAAT) levels after infusion of AT-SCs overexpressing hAAT. Cells were transplanted into syngeneic mice after CCl₄-induced hepatic injury. Luciferase bioluminescence signals and serum hAAT levels were measured at different time points after transplantation and demonstrate persistence of transplanted cells for up to 2 months after administration. These data, along with immunohistochemical analysis, suggest engraftment and repopulation of injured livers by transplanted AT-SCs. Moreover, by transcriptional targeting using cellular tissue-specific regulatory sequences we confirmed that AT-SCs differentiate towards a hepatogenic-like phenotype in vitro and in vivo. Additionally, in transplanted cells re-isolated from recipient animals ' livers we detected activation of the alpha-fetoprotein (AFP) promoter. This promoter is normally transcriptionally silenced in adult tissues but can be re-activated during liver regeneration, suggesting commitment towards hepatogenic-like differentiation of engrafted cells in vivo. Our data support AT-SC cell-mediated gene therapy as an innovative therapeutic option for disorders of liver metabolism.
    Cell Transplantation 03/2012; 21(9). DOI:10.3727/096368911X637452 · 3.57 Impact Factor