August 1999
Volume 40, Issue 9
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Retina  |   August 1999
Tracking RPE Transplants Labeled by Retroviral Gene Transfer with Green Fluorescent Protein
Author Affiliations
  • Chi-Chun Lai
    From the Departments of Ophthalmology and
  • Peter Gouras
    From the Departments of Ophthalmology and
  • Ken Doi
    From the Departments of Ophthalmology and
  • Fang Lu
    From the Departments of Ophthalmology and
  • Hild Kjeldbye
    From the Departments of Ophthalmology and
  • Steven P. Goff
    Biochemistry, and Molecular Biophysics, Columbia University, Howard Hughes Medical Institute, New York, New York;
  • Robert Pawliuk
    Genetix Pharmaceuticals, Inc., Cambridge, Massachusetts; and
  • Philippe Leboulch
    Harvard Medical School; Massachusetts Institute of Technology;
  • Stephen H. Tsang
    Biochemistry, and Molecular Biophysics, Columbia University, Howard Hughes Medical Institute, New York, New York;
Investigative Ophthalmology & Visual Science August 1999, Vol.40, 2141-2146. doi:
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      Chi-Chun Lai, Peter Gouras, Ken Doi, Fang Lu, Hild Kjeldbye, Steven P. Goff, Robert Pawliuk, Philippe Leboulch, Stephen H. Tsang; Tracking RPE Transplants Labeled by Retroviral Gene Transfer with Green Fluorescent Protein. Invest. Ophthalmol. Vis. Sci. 1999;40(9):2141-2146.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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. GFP-expressing 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.

The 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% CO2, 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 5′ 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 105 to 106 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 CaPO4 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 GFP-transduced cells. The lentiviral titers were determined by infection of 293 cells seeded in 6-well plates at 1 × 105 cells per well the day before infection with serial dilution of concentrated viral stock in the presence of 8 μg/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 108 to 109 infectious units/ml. 
In Vitro Transfection
For viral transduction, primary cultures were dissociated into cell suspensions and subcultured in 6-well plates containing approximately 105 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 μg/ml polybrene, and a viral titer of 105 to 107 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 × 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 100μ 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 μl of cell suspension containing approximately 105 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. 
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 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. Figure 1 shows heavily pigmented stationary cells within a patch culture and more lightly pigmented dividing cells spreading our from this patch, viewed by both fluorescent and transmitted white light, 6 weeks after exposure to Lentivirus. Figure 1B shows only the fluorescence of the same area. Green fluorescence can be seen in both the stationary and dividing cells. Figure 1C shows a heavily pigmented patch of stationary cells, which do not show any fluorescence (upper left). These cells appear identical with controls that have not been exposed to the virus. The fluorescence seen in this culture (Fig. 1D) occurs only at the edge of the patch and in the lightly pigmented cells migrating away from the patch. Subcultured cells continue to express GFP. The overall level of expression remained relatively stable for a 3-month period of observation. Immunohistochemistry confirmed the presence of GFP protein in the cultured RPE. Figure 2 shows the fraction of HFRPE cells expression detectable GFP fluorescence in vitro for 2 months. Approximately 30% of the cultured cells showed expression of high level of GFP fluorescence within the first week after exposure to the virus. This fraction slowly increased during the first month to reach approximately 45%. 
Figure 3 shows an HFRPE transplant in the subretinal space of pigmented rabbit retina at 1 week after surgery by visible (red) (A) and infrared (B) illumination and fluorescence SLO (C). The transplant was better seen as a dark subretinal structure by infrared than by visible illumination; even its overall thickness can be estimated by its absorption of infrared light. Fluorescence revealed the GFP-expressing cells within the transplant. The dimensions of these bright structures with relatively sharp outlines approximated those of either single cells or small groups of cells. The fluorescent cells are congruent with the dark subretinal transplant seen by infrared illumination. The transplanted cells fill the area of the original bleb detachment visible by a dim demarcation line seen better by visible than infrared illumination. 
Figure 4 shows a transplant site at 5 days after surgery by infrared (A) and fluorescence SLO (B, C, D). At 5 days, many GFP-fluorescing cells can be seen in the center of the transplant (Fig. 3B) . At 9 days after surgery, many fluorescent cells have disappeared (Fig. 3C) . At 2 weeks very few fluorescent cells remain. After 3 to 4 weeks, GFP fluorescence could not be detected in these transplants. There was a steady decrease in the amount of GFP fluorescence in these transplants with time after transplantation surgery. 
Histology revealed GFP fluorescent cells in the subretinal space within 1 week after transplantation surgery (Fig. 1E) . Between 1 and 3 weeks after surgery, the transplant site showed signs of rejection (Fig. 1F) , and GFP-fluorescing cells became difficult to find. There was a dense concentration of monocytic cells in the choroid adjacent to the transplant site. The high concentration of inflammatory cells appeared to fill and/or compress the choroidal vessels and the choriocapillaries. Despite this intense inflammatory response in the choroid, there was no evidence of staining or leakage of dye during fluorescein and ICG angiography performed repeatedly in every rabbit. 
Discussion
Replication deficient retroviruses can introduce a foreign gene into HFRPE in vitro. This gene remains able to express a unique protein, in this case GFP, at a relatively constant rate for long periods of time, at least 3 months. Lentivirus was a more effective vector than MoMLV because it transduced both nondividing as well as dividing cells. MoMLV only transduced the latter. RPE patch cultures provide an easily identifiable and discrete group of cells that show no evidence of division. Cells within such patches retain the same appearance and pigmentation and do not increase in number for months. Dividing cells are easy to identify by their progressive loss of pigmentation, their continuous migration away from the edge of the patch, and their increase in numbers. 7 Therefore, human fetal RPE provides a good system to examine the transduction of dividing versus stationary, presumably nondividing, cells in vitro. 
The variation in GFP fluorescence among the cells may be due to different sites of chromosomal integration, assuming this occurs. It also may be due to multiple integration and/or expression sites within the same cell. The fact that GFP expression is continuously maintained after subculturing and repeated cell division suggests that chromosomal integration has occurred. This can be better tested by Southern blot analysis or polymerase chain reaction of the GFP gene in genomic DNA. 
GFP exhibits a strong, nonquenchable fluorescence that is easy to monitor by SLO viewing. Single transplanted cells or small groups of cells can be distinguished and followed noninvasively and long term. In our experiments, GFP-expressing HFRPE was transplanted into the subretinal space of rabbits, therefore, as xenografts. This led to rejection usually within 1 to 3 weeks after transplantation surgery. GFP-fluorescent cells were identifiable within the subretinal space in relatively large numbers within the first week after transplantation, but their numbers began to diminish as rejection progressed. The changes in GFP fluorescence followed the time course of the rejection process observed histologically. Therefore, GFP expression provided an in vivo monitor of the viability and gene expression of transplanted RPE. 
The fact that a substantial number of cells do not show fluorescence reduces the sensitivity of the technique. This can be improved by FACS just before transplantation. On the other hand, GFP fluorescence appears to be specific for the transplanted cells because all trace of its fluorescence disappears after the transplant is rejected. Host rejection has prevented testing the long-term expression of these cells in vivo. This can be done by using allografts or homografts rather than xenografts in the future. 
We found that GFP is an excellent monitor of not only the viability but also the function of the transplanted RPE, as manifest by gene expression and protein synthesis. By using this marker, one can track the presence of cells placed in the subretinal space and also monitor whether they are expressing a potentially therapeutic gene linked in tandem to the expression of GFP. The fact that RPE can be cultured easily provides a way to optimize the expression of a particular gene in vitro before it is introduced into the subretinal space. The question of rejection could of course be eliminated by using autografts such as cultured iris pigment epithelium from the same subject who is the target of such ex vivo form of gene therapy. Future engineering of RPE cells by retroviral vectors will undoubtedly augment their use in transplantation. In addition a marker that can be used to identify transplanted cells in the retina ophthalmoscopically will facilitate determining whether certain RPE allografts can survive in the subretinal space of human subjects without immunosuppression. 8  
 
Figure 1.
 
Color photographs of cultured HFRPE transfected with the GFP gene by Lentivirus 6 weeks previously. (A) An HFRPE patch culture (on the left) spreading out across the culture plate (on the right) photographed by both fluorescent and transmitted white light. Strong green fluorescence is visible on the left. (B) The same area as (A), photographed by fluorescence only. Both stationary (left) and dividing (right) cells show GFP fluorescence. (C) A HFRPE patch culture that shows no fluorescence (upper left) except along its border with cells migrating from its edge. (D) The same area as (C), photographed by fluorescence only. There is no fluorescence in the upper left, which appears identical with control cultures never exposed to the virus. GFP fluorescence is seen only along the edge of this patch and in the dividing cells (right). (E) GFP fluorescent HFRPE transplanted to the subretinal space of rabbit 1 week earlier. (F) Histologic section, stained with toluidine blue, showing cellular inflammation in a transplant site at 9 days after surgery. Asterisks, RPE cells with melanin; curved arrows, monocytes; straight arrows, rare choriocapillary.
Figure 1.
 
Color photographs of cultured HFRPE transfected with the GFP gene by Lentivirus 6 weeks previously. (A) An HFRPE patch culture (on the left) spreading out across the culture plate (on the right) photographed by both fluorescent and transmitted white light. Strong green fluorescence is visible on the left. (B) The same area as (A), photographed by fluorescence only. Both stationary (left) and dividing (right) cells show GFP fluorescence. (C) A HFRPE patch culture that shows no fluorescence (upper left) except along its border with cells migrating from its edge. (D) The same area as (C), photographed by fluorescence only. There is no fluorescence in the upper left, which appears identical with control cultures never exposed to the virus. GFP fluorescence is seen only along the edge of this patch and in the dividing cells (right). (E) GFP fluorescent HFRPE transplanted to the subretinal space of rabbit 1 week earlier. (F) Histologic section, stained with toluidine blue, showing cellular inflammation in a transplant site at 9 days after surgery. Asterisks, RPE cells with melanin; curved arrows, monocytes; straight arrows, rare choriocapillary.
Figure 2.
 
The relationship between the fraction of cultured HFRPE-expressing GFP fluorescence at different times after transduction with Lentivirus. Each data point is the average of measurements made in three different areas of 10 separate cultures; the vertical lines show the standard errors of the mean.
Figure 2.
 
The relationship between the fraction of cultured HFRPE-expressing GFP fluorescence at different times after transduction with Lentivirus. Each data point is the average of measurements made in three different areas of 10 separate cultures; the vertical lines show the standard errors of the mean.
Figure 3.
 
A transplant site in rabbit retina seen by SLO one week after surgery. The transplant site is the oval-like structure on the left with its upper edge just under the myelinated optic nerve fibers (seen best in A). (A) Red light image shows the transplant mainly by a light border that surrounds it. (B) Infrared image shows the transplant as a darker granular structure that stands out in the lighter normal retina. (C) Fluorescence image that reveals the GFP-positive cells in the transplant.
Figure 3.
 
A transplant site in rabbit retina seen by SLO one week after surgery. The transplant site is the oval-like structure on the left with its upper edge just under the myelinated optic nerve fibers (seen best in A). (A) Red light image shows the transplant mainly by a light border that surrounds it. (B) Infrared image shows the transplant as a darker granular structure that stands out in the lighter normal retina. (C) Fluorescence image that reveals the GFP-positive cells in the transplant.
Figure 4.
 
SLO photographs of an HFRPE xenograft in rabbit retina observed with infrared (A) and fluorescence (B) illumination at 5 days after surgery. The appearance of the transplant by fluorescence illumination at 9 days (C) and 2 weeks (D) after surgery shows a progressive diminution in the number of GFP fluorescent cells.
Figure 4.
 
SLO photographs of an HFRPE xenograft in rabbit retina observed with infrared (A) and fluorescence (B) illumination at 5 days after surgery. The appearance of the transplant by fluorescence illumination at 9 days (C) and 2 weeks (D) after surgery shows a progressive diminution in the number of GFP fluorescent cells.
The authors thank Inder M. Verma of the Salk Institute, La Jolla, California, for his generous contribution of the plasmids for establishing the Lentivirus vector, and Dorothy Warburton and Karen Weidenheim for their assistance. 
Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell Comp Physiol.. 1962;59:223–227. [CrossRef] [PubMed]
Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green fluorescent protein. Gene. 1992;111:229–233. [CrossRef] [PubMed]
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Lai C, Pawliuk R, Gouras P, Tsang S, Lu F, Doi K, et al. Genetically engineered human RPE transplants express green fluorescent protein in the subretinal space[ARVO Abstract]. Invest Ophthalmol Vis Sci. 1998;39(4):S19. Abstract nr 73.
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Gouras P, Cao H, Sheng Y, Tanabe T, Efremova Y, Kjeldbye H. Patch culturing and transfer of human fetal retinal epithelium. Graefes Arch Clin Exp Ophthalmol. 1994;232:599–607. [CrossRef] [PubMed]
Algvere P, Berglin L, Gouras P, Sheng Y, Dafgard Kopp E. Transplantation of RPE in age-related macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch Clin Exp Ophthalmol. 1997;235:149–158. [CrossRef] [PubMed]
Figure 1.
 
Color photographs of cultured HFRPE transfected with the GFP gene by Lentivirus 6 weeks previously. (A) An HFRPE patch culture (on the left) spreading out across the culture plate (on the right) photographed by both fluorescent and transmitted white light. Strong green fluorescence is visible on the left. (B) The same area as (A), photographed by fluorescence only. Both stationary (left) and dividing (right) cells show GFP fluorescence. (C) A HFRPE patch culture that shows no fluorescence (upper left) except along its border with cells migrating from its edge. (D) The same area as (C), photographed by fluorescence only. There is no fluorescence in the upper left, which appears identical with control cultures never exposed to the virus. GFP fluorescence is seen only along the edge of this patch and in the dividing cells (right). (E) GFP fluorescent HFRPE transplanted to the subretinal space of rabbit 1 week earlier. (F) Histologic section, stained with toluidine blue, showing cellular inflammation in a transplant site at 9 days after surgery. Asterisks, RPE cells with melanin; curved arrows, monocytes; straight arrows, rare choriocapillary.
Figure 1.
 
Color photographs of cultured HFRPE transfected with the GFP gene by Lentivirus 6 weeks previously. (A) An HFRPE patch culture (on the left) spreading out across the culture plate (on the right) photographed by both fluorescent and transmitted white light. Strong green fluorescence is visible on the left. (B) The same area as (A), photographed by fluorescence only. Both stationary (left) and dividing (right) cells show GFP fluorescence. (C) A HFRPE patch culture that shows no fluorescence (upper left) except along its border with cells migrating from its edge. (D) The same area as (C), photographed by fluorescence only. There is no fluorescence in the upper left, which appears identical with control cultures never exposed to the virus. GFP fluorescence is seen only along the edge of this patch and in the dividing cells (right). (E) GFP fluorescent HFRPE transplanted to the subretinal space of rabbit 1 week earlier. (F) Histologic section, stained with toluidine blue, showing cellular inflammation in a transplant site at 9 days after surgery. Asterisks, RPE cells with melanin; curved arrows, monocytes; straight arrows, rare choriocapillary.
Figure 2.
 
The relationship between the fraction of cultured HFRPE-expressing GFP fluorescence at different times after transduction with Lentivirus. Each data point is the average of measurements made in three different areas of 10 separate cultures; the vertical lines show the standard errors of the mean.
Figure 2.
 
The relationship between the fraction of cultured HFRPE-expressing GFP fluorescence at different times after transduction with Lentivirus. Each data point is the average of measurements made in three different areas of 10 separate cultures; the vertical lines show the standard errors of the mean.
Figure 3.
 
A transplant site in rabbit retina seen by SLO one week after surgery. The transplant site is the oval-like structure on the left with its upper edge just under the myelinated optic nerve fibers (seen best in A). (A) Red light image shows the transplant mainly by a light border that surrounds it. (B) Infrared image shows the transplant as a darker granular structure that stands out in the lighter normal retina. (C) Fluorescence image that reveals the GFP-positive cells in the transplant.
Figure 3.
 
A transplant site in rabbit retina seen by SLO one week after surgery. The transplant site is the oval-like structure on the left with its upper edge just under the myelinated optic nerve fibers (seen best in A). (A) Red light image shows the transplant mainly by a light border that surrounds it. (B) Infrared image shows the transplant as a darker granular structure that stands out in the lighter normal retina. (C) Fluorescence image that reveals the GFP-positive cells in the transplant.
Figure 4.
 
SLO photographs of an HFRPE xenograft in rabbit retina observed with infrared (A) and fluorescence (B) illumination at 5 days after surgery. The appearance of the transplant by fluorescence illumination at 9 days (C) and 2 weeks (D) after surgery shows a progressive diminution in the number of GFP fluorescent cells.
Figure 4.
 
SLO photographs of an HFRPE xenograft in rabbit retina observed with infrared (A) and fluorescence (B) illumination at 5 days after surgery. The appearance of the transplant by fluorescence illumination at 9 days (C) and 2 weeks (D) after surgery shows a progressive diminution in the number of GFP fluorescent cells.
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