Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein

PURPOSE. To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO). METHODS. Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically. RESULTS. Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector traduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFPexpressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically. CONCLUSIONS. Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space. (Invest Ophthalmol Vis Sci. 1999;40:2141-2146 T he green fluorescent protein (GFP) gene has been derived from the bioluminescent jelly fish, Aequorin victoria. This protein fluoresces green light when excited by blue or ultraviolet light. The cloning of this gene and the demonstration that it can be expressed in other organisms provides a useful way to select and follow cells exhibiting specific gene expression, [1][2][3] especially in a transparent structure such as the eye. 4 We have used replication-deficient retroviruses to transduce cultured human fetal retinal pigment epithelium (HFRPE) with the gene encoding GFP. We followed the expression of this protein in vitro by fluorescence microscopy and in vivo after transplantation to the subretinal space of rabbits by scanning laser ophthalmoscopy (SLO). We demonstrated that retroviral transduction is effective, stable, and long-lasting in vitro. It allows transplanted RPE to be monitored in the subretinal space and provides a noninvasive indicator of the time course of rejection. An abstract on some of this research has been published. 5 METHODS Culturing of RPE Donor tissue was obtained from human fetal eyes, 16 to 20 weeks of gestational age. Informed consent was obtained for the use of this tissue before abortion and institutional approval was granted through an agreement between Albert Einstein College of Medicine, the source of the tissue, and Columbia University. The eye bulbs were washed externally with 70% alcohol and then with phosphate-buffered saline (PBS). The eyes were put into our standard RPE culture medium, Dulbecco's modified Eagle's medium with 4.5 g/l glucose supplemented with 20% fetal calf serum (Hyclone, Logan, Utah), 2 mM L-glutamine and penicillin (50 unit/ml)/streptomycin (50 mg/ml) (Gibco, Grand Island, NY). The anterior segment with lens, vitreous, and neural retina was removed. The posterior segment was sliced into quadrants, and RPE patches were separated gently from Bruch's membrane and choroid, using fine forceps and microscopic viewing. A distinct cleavage plane is identifiable between the taut monolayer patch of RPE and the adjacent choroid so that an isolated sheet of RPE can be pulled off. Each sheet was placed in a separate culture plate. The edges of the sheet were pressed onto the surface of the plate with the tip of a 26-gauge needle. The cultures were maintained at 37°C in an incubator with a humidified atmosphere of 95% air/5% CO 2 , fed every 3 to 4 days, and examined almost daily. To obtain cell suspensions, we washed the cells with PBS three times and exposed them to 2.5% trypsin in Hank's solution with EDTA without Ca and Mg (Gibco) for 10 minutes at 37°C. The monolayer was triturated into single cells or clusters of cells by repeated pipetting. The concentration of cells in a suspension was determined with a hemocytometer. The cells were either used for transplantation or subcultured. Preparation of Virus Stocks For the Moloney vector a DNA construct was generated consisting of the humanized red-shifted GFP (EGFP) under the translational control of an Internal Ribosome Entry Site from the encephalomyocarditis virus (EMCV-IRES), flanked by long terminal repeat (LTR) of Moloney murine leukemia virus (MoMLV). These viral sequences include the two LTRs, and the two sites for initiation of viral DNA synthesis (the primer binding site for initiation of minus-strand DNA synthesis and the polypurine tract for initiation of plus-strand synthesis). They also include the RNA packaging signal, termed the Psi region, near the 5Ј end of the genome. The construct was then introduced into AM 12 packaging cells that express the viral proteins required for the assembly of a virion particle. The viral RNA was transcribed from a transfected plasmid and selectively packaged into viral particles produced by the packaging cells. The virions were collected from the culture medium, purified, and concentrated as needed. To transduce the gene to RPE, the virus was applied directly to the target cells. Typical titers were 10 5 to 10 6 infectious units/ml. For the lentiviral vector, human immunodeficiency virus (HIV)-based preparations were generated by cotransfection of human kidney-derived 293T cells by three plasmids using the CaPO 4 method. 6 The packaging construct contained the cytomegalovirus promoter and the insulin polyadenylation signal to express all the viral proteins in trans, except the envelope and Vpu. 6 The second plasmid provided a vector with all the cis-acting elements that allow transfer and integration into the target cell. In this transducing vector, an expression cassette with the Rev responsive element (RRE) and the cytomegalovirus promoter are used to direct the expression of GFP. 6 The third plasmid provides the envelope protein from the vesicular stomatitis virus glycoprotein to enhance the viral stability and the range of possible target cells. 6 The titer of the HIV vector was determined by a fluorescent activated cell sorter (FACStar plus; Becton Dickinson, Mountain View, CA) scanning GFPtransduced cells. The lentiviral titers were determined by infection of 293 cells seeded in 6-well plates at 1 ϫ 10 5 cells per well the day before infection with serial dilution of concentrated viral stock in the presence of 8 g/ml of polybrene (Aldrich, Milwaukee, WI). After overnight incubation, the cell culture medium was changed, and the cells were incubated further for 2 days. GFP fluorescent cells were identified by fluorescent microscopy and/or the FACS. Typical titers were 10 8 to 10 9 infectious units/ml. In Vitro Transfection For viral transduction, primary cultures were dissociated into cell suspensions and subcultured in 6-well plates containing approximately 10 5 cells/well. This promotes cell division and augments the total number of cells available. After 24 hours in standard RPE culture medium, the medium was replaced with the viral solution, consisting of Hepes buffer with 20% fetal calf serum, 2 mM L-glutamine, 8 g/ml polybrene, and a viral titer of 10 5 to 10 7 infectious units/ml before concentration. This solution was replaced with fresh viral solution every 6 hours for 48 hours. After 48 hours, this solution was replaced with standard RPE medium, and the cultures were allowed to reach confluency, examined by fluorescence microscopy, and used for transplantation. For comparing viral transduction of stationary versus dividing cells, the virus was introduced directly into the primary culture containing the original patch of heavily pigmented cells, surrounded by an expanding population of dividing cells, the size of which depended on the age of the culture. To determine the fraction of cells expressing GFP, the number of GFP fluorescent cells and the total number of cells were counted within defined areas, 0.4 ϫ 0.8 mm, in the culture plate. All cells that showed green fluorescence were considered to be expressing GFP. We examined cells in the same areas in three different parts of each culture plate, the patch that contained stationary pigmented cells only, the edge of the patch where cells were migrating and entering into cell division, and the growing margin of the culture, which contained many fewer pigmented, dividing cells. We measured these same areas repeatedly in 15 different cultures, weekly for 3 weeks; 5 cultures were measured for 6 weeks, and 1 culture for 3 months. In one case, we dissociated a primary culture that we had examined for 3 months and replated the cells to follow GFP fluorescence after repeated cell division. Transplantation Thirty adult pigmented rabbits received subretinal transplants placed within small bleb detachments just below the myelinated region of the optic nerve. Bleb detachments also were formed in two rabbits with saline alone. Each animal was anesthetized with sodium pentobarbital (25 mg/kg, intramuscularly) and xylazine (10 mg/kg, intramuscularly). The pupil was dilated with 2% cyclopentolate and 2.5% neosynephrine. A lid speculum was used to keep the eye open and occasionally a canthotomy also was performed. A conjunctival flap was formed at the limbal region, and a sclerotomy made approximately 3 mm behind the limbus. A glass pipette with a tip diameter of 80 to 100 m, connected to a 1-ml syringe and filled with balanced salt solution (BSS) was introduced into the vitreal cavity. Using a corneal contact lens and a surgical microscope the pipette was directed to the retinal surface. At the surface of the retina a jet stream of BSS was slowly injected through the neural retina to produce a small bleb detachment. A second similar pipette was used to suck up a pellet of a concentrated solution of GFP-expressing HFRPE cells from the bottom of an Eppendorf tube. The cell suspension was obtained by rinsing a culture three times with PBS and then dissociating the cells with 0.05% trypsin for 5 minutes at 37°C. The cells were washed with PBS and centrifuged. The pellet was resuspended in 0.5 ml BSS, put into an Eppendorf tube, centrifuged at 1000 rpm for 2 minutes, and stored at 4°C. The cells were used within 2 hours of preparation. Approximately 10 l of cell suspension containing approximately 10 5 cells was introduced into the bleb detachment, either through the same retinotomy or through a second one; the latter method was preferable because it minimized any reflux of transplant cells into the vitreous. A small air bubble separated the suspension from the BSS solution in the pipette. The bubble also was introduced into the bleb detachment to prevent efflux of the transplant cells into the vitreous. The air bubble disappeared in 24 hours. After the pipette was removed, the sclera and conjunctiva were sutured with 9-0 nylon. Reports IOVS, August 1999, Vol. 40, No. 9 Downloaded from iovs.arvojournals.org on 06/30/2019 Retinal Examination Rabbits were examined 1 day after surgery, weekly for 8 weeks, and monthly thereafter by indirect ophthalmoscopy, SLO (Rodenstock, Munich, Germany) and sometimes by contact lens biomicroscopy. The SLO provided infrared (780 nmoles), He-Neon red (633 nmoles), argon green (514 nmoles), and blue (488 nmoles) illumination. We examined retinal fluorescence with argon blue illumination and a fluorescein barrier filter. We graded the fluorescence using a scale of 0 to 4 (0, no fluorescence,; 1, just detectable; 2, distinct; 3, strong; 4, very strong). Fluorescein and indocyanine green (ICG) angiography were performed simultaneously with an SLO double-detection system that was able to detect fluorescein and ICG simultaneously. Angiography was performed August 1999, Vol. 40, No. 9 Reports IOVS, weekly for 2 to 3 weeks and monthly thereafter. The dyes were injected into an ear vein in one bolus containing 0.2 ml fluorescein (100 mg/ml) and 0.7 ml ICG (4.2 mg/ml). Histology After the rabbit was euthanatized, the eyes were enucleated, punctured with a 20 gauge needle at several places near the limbus to facilitate diffusion, and immersed in a solution of either 3% glutaraldehyde or 4% paraformaldehyde in PBS at pH 7.2 for 24 to 48 hours at 4°C. The eyes then were washed with PBS and dissected with the aid of a microscope. The transplant site was located, examined, and cut out with its orientation marked so that the site could be reached with minimal sectioning. For Epon embedding, glutaraldehyde-fixed segments were postfixed with 1% osmic acid and dehydrated with ethanol. Sections were cut semi-serially and examined by light microscopy; selected areas were examined by electron microscopy. For cryosectioning paraformaldehyde-fixed segments were immersed in OCT compound (Miles, Elkhart, IN) and frozen by dry ice. Cryosectioning was performed on a Leica 1850 cryotome (Leica Instruments, Nusslach, Germany). Sections were mounted on gelatinized glass slides with fluoromount-G. GFP polyclonal antibody (diluted 1:100; Clontech Laboratories, Palo Alto, CA) was used for immunocytochemistry. Cultured RPE cells not exposed to the virus were used as a negative control. RESULTS GFP fluorescence was detectable in cultured HFRPE within 5 days after being exposed to the retrovirus. The MoMLV only transduced dividing cells that occurred along the edge of patch cultures spreading out centrifugally over the culture plate. 7 The lentivirus transduced both stationary and dividing cells