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. 2008:14:2117-25.
Epub 2008 Nov 26.

Feline immunodeficiency virus-mediated long-term transgene expression in undifferentiated retinal progenitor cells and its downregulation in differentiated cells

Branislava Janic  1 , Xuxiang Zhang, Wei Li
Affiliations

Feline immunodeficiency virus-mediated long-term transgene expression in undifferentiated retinal progenitor cells and its downregulation in differentiated cells

Branislava Janic et al. Mol Vis. 2008.

Abstract

Purpose: Lentivirus-mediated gene transfer is an important approach to modify the function of progenitor cells in ex vivo gene therapy, but may be susceptible to downregulation due to transcriptional silencing. The purpose of this study was to analyze the stability of lentivirus-mediated transgene expression in undifferentiated and differentiated retinal progenitor cells (RPCs), and to characterize the effect of lentivirus transduction on RPC differentiation in vitro.

Methods: RPCs derived from postnatal day 1 mice were expanded in defined serum-free culture medium and transduced with nonprimate lentiviral vector of feline immunodeficiency virus (FIV) expressing yellow fluorescent protein (YFP) reporter. Long-term expression of YFP in undifferentiated and differentiated RPCs was analyzed. Expression of various markers for RPCs and differentiated cells was analyzed by immunochemical staining in lentivirus-transduced and control RPCs. Differentiated postmitotic cells were revealed by negative labeling of bromodeoxyuridine (BrdU).

Results: FIV transduction induced long-term expression of YFP reporter in RPCs for up to 53 days (10 passages) with no sign of decrease in expression level. FIV transduction did not alter the expression profile of various markers in retinal spheres, including nestin, microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP), and opsin. However, YFP expression was downregulated in differentiated BrdU-negative postmitotic cells.

Conclusions: FIV-mediated long-term expression of transgene in undifferentiated RPCs is downregulated upon their differentiation. Thus, lentivirus-mediated ex vivo modulation should be cautiously analyzed for transgene expression not only in undifferentiated RPCs, but also in differentiated postmitotic cells.

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Figures

Figure 1
Figure 1
Lentivirus transduction of retinal progenitor cells. A: Schematic representation of feline immunodeficiency virus (FIV) vector. Abbreviations are: LTR, FIV long-terminal repeat; CMV, human cytomegalovirus promoter, and YFP, yellow fluorescent protein. B: In vitro live fluorescence image of retinal progenitor cells (RPCs) spheres for yellow fluorescent protein (YFP) expression (top, fluorescence field; bottom, bright field). RPCs were transduced with FIV-YFP and analyzed for YFP expression at day 8 post-transduction with an Olympus IX50 inverted fluorescence microscope. C: Flow cytometry analysis of YFP expression in RPCs. Flow cytometric histogram of control cells (filled gray histogram) and FIV-YFP transduced cells (open black line) from representative experiment is shown. At least 5,000 gated cells were analyzed for YFP expression. The majority of FIV-transduced RPCs (97.5%) were YFP positive.
Figure 2
Figure 2
Nestin expression in FIV-transduced retinal progenitor cells. A: Immunocytochemistry for nestin expression (red signal) in retinal progenitor cell (RPC) spheres cultured under proliferating conditions. The nuclei of all cells were stained with DAPI (blue signal) and analyzed using a Zeiss Axiovert 200 m fluorescence microscope. B: Quantitative analysis of nestin-positive cells in feline immunodeficiency virus (FIV) for yellow fluorescent protein (YFP)-infected and noninfected RPCs. The percentage of nestin-expressing cells was determined by counting at least 100 cells in three independent experiments. Results are expressed as mean±SEM; p<0.05 (Student’s t-test; FIV-YFP versus control).
Figure 3
Figure 3
Long-term expression of YFP reporter in FIV-transduced RPCs. Dissociated RPCs were transduced with feline immunodeficiency virus (FIV) expressing yellow fluorescent protein (YFP) and plated at a low density of 10 cells/μl in the complete growth medium to generate clonal spheres. Cells were monitored, and images captured at days 1, 4, and 7 post-dissociation by the inverted fluorescence microscope to detect the presence of secondary RPC spheres. Long-term expression of YFP reporter was still detected after 10 passages. Shown are live-phase contrast cell images (fluorescence and bright field) from the representative culture.
Figure 4
Figure 4
Multipotentiality. A: Retinal progenitor cells (RPCs) transduced with feline immunodeficiency virus (FIV) for yellow fluorescent protein (YFP) were cultured under differentiated conditions for 14 days to give rise to neuronal and glial cell types. Cells that stained positive for microtubule-associated protein 2 (MAP-2), opsin, and glial fibrillary acidic protein (GFAP) were analyzed by confocal microscopy, and signaling displayed in red. Green signal was YFP, and blue signal was DAPI staining for nuclei. B: Quantitative analysis of cells positive for MAP-2, opsin, and GFAP in RPC group infected with or without FIV-YFP. At least 100 cells were counted from three independent experiments. Results are expressed as mean±SEM; p>0.05 (Student’s t-test; FIV-YFP versus control).
Figure 5
Figure 5
YFP expression in differentiated retinal progenitor cells. A: Live images of FIV-YFP infected cells cultured for 14 days under differentiated conditions from a representative experiment. The cells have adopted a variety of neuronal cell morphologies and have downregulated the expression of YFP (arrows). B: Immunocytochemistry of cells labeled with BrdU (red signal) and cultured under differentiating conditions. BrdU-negative cells downregulated the expression of YFP (arrows). The nuclei of all cells were stained with DAPI (blue signal). The results were examined using a Zeiss Axiovert fluorescence microscope, and images from the representative experiments are shown.
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