Transplantation of human fetal retinal pigment epithelium rescues photoreceptor cells from degeneration in the Royal College of Surgeons rat retina

Department of Neurobiology, University of Rochester School of Medicine, NY 14642, USA.
Investigative Ophthalmology &amp Visual Science (Impact Factor: 3.4). 02/1996; 37(1):204-11. DOI: 10.1097/00006982-199717050-00030
Source: PubMed


The Royal College of Surgeons (RCS) rat suffers from a well-characterized, early-onset, and relentless form of photoreceptor cell degeneration. It has been shown that allografts of retinal pigment epithelial cells from normal perinatal rats have rescue effects in this condition. In preparation for human application, the authors determined whether human fetal retinal pigment epithelium (RPE) grafts have a photoreceptor rescue effect in RCS dystrophic rat retinas.
Sheets of RPE from human fetal eyes (10 to 16 weeks gestational age) were isolated according to the authors' recently described method. Fragments of the RPE sheets were transplanted to the subretinal space within the superior hemisphere. Transplants were performed within the superior equatorial region of five dystrophic RCS rats, one eye per animal. A similar volume of vehicle was injected into the subretinal space of five age-matched control rats, again one eye per rat. All rats were immunosuppressed with daily injections of cyclosporine. Using light microscopy, photoreceptor cell nuclear profiles of superior equatorial (SE) and inferior equatorial (IE) regions of transplanted and sham-injected control animals were counted.
Four weeks after transplantation, a dramatic rescue effect was observed. Microscopically, presumptive donor RPE cells were seen as single pigmented cells and as cell clusters in the subretinal space. An outer nuclear layer three to four profiles thick was present in the area of the RPE transplant but was nearly absent in the rest of the retina, as well as in the retinas of control rats. The number of photoreceptor nuclear profiles per 100 microns was 34.7 +/- 2.2 (mean +/- SEM) in the SE region of transplanted rats and 3.5 +/- 1.4 in the same region of sham-injected rats. There were 3.0 +/- 1.0 photoreceptor nuclear profiles in the IE region of transplanted rats and 3.5 +/- 1.2 in the IE region of sham-injected eyes. No evidence of graft rejection was seen.
This study provides the first indication that transplanted human fetal RPE cells are able to rescue photoreceptor cells in a model of hereditary retinal degeneration.

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    • "Several types of RPE cells have been studied and used for transplantation experiments in animal models (Pfeffer and Philp, 2014), including cell lines (Coffey et al., 2002; Wang et al., 2005), fetal (Little et al., 1996, 1998) and adult human RPE cells (Castillo et al., 1997) as well as stem cell-derived RPE cells (Lund et al., 2006; Vugler et al., 2008; Carr et al., 2009; Lu et al., 2009). Besides human tissue-derived cells, RPE cells from various animal species have been used for the transplantation into animal models (Li and Turner, 1988). "
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    ABSTRACT: Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. Recent approaches to cure this disease include the transplantation of stem cell-derived RPE cells. However, deriving RPE cells from stem cells takes several weeks. AMD is age-related and therefore modelling and studying the disease in vitro using adult RPE cells seems more appropriate. Moreover, to study the disease in animal models, the use of cells derived from the species makes immunosuppression unnecessary. For most transplantation studies cells derived from young animals have been used. However, the There are only three protocols in the current literature that use adult animal tissue to isolate RPE cells. These protocols are not optimized for RPE cell culturing, and they did not yield sufficient numbers of RPE cells in our hand. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the isolated RPE cells bound to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro transplantation experiments to study retinal diseases.
    Frontiers in Neuroscience 01/2015; · 3.66 Impact Factor
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    • "This pathology is autologous to a human form of RP (Gal et al., 2000), and by virtue of the primary defect being in the RPE cells, it may serve as an analogous model of some aspects of AMD. A series of studies has shown that subretinal injections of cells obtained from a range of human and animal cell lines can limit rod and cone cell death in RCS rats (Arnhold et al., 2006; Gamm et al., 2007; Huang et al., 2006; Inoue et al., 2007; Lawrence et al., 2000, 2004; Lin, Fan, Sheedlo , Aschenbrenner, & Turner, 1996; Little et al., 1996; Lund et al., 2001; Lund, Wang, Klimanskaya, Holmes, & Ramos-Kelsey, 2006; Mizumoto, Mizumoto, Shatos, Klassen, & Young, 2003; Pinilla, Cuenca, Sauvé, Wang, & Lund, 2007; Rezai, Kohen, Wiedemann, & Heimann, 1997; Schraermeyer et al., 2001; Schraermeyer, Kociok, & Heimann, 1999; Seiler, Sagdullaev, Woch, Thomas, & Aramant, 2005; Thumann, Salz, Walter, & Johnen, 2009; Wang, Lu, & Lund, 2005; Wang, Lu, Wood, & Lund, 2005; Wojciechowski, Englund, Lundberg, Wictorin, & Warfvinge, 2002). Moreover, electrophysiological (Lund et al., 2001; Sauvé, Girman, Wang, Keegan, & Lund, 2002; Sauvé, Klassen, Whiteley, & Lund, 1998; Sauvé, Lu, & Lund, 2004) and psychophysical (McGill, Douglas, Lund, & Prusky, 2004; McGill, Lund, Douglas, Wang, Lu, & Prusky, 2004) assessments have shown that some level of visual function can be preserved in such treated animals. "
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    ABSTRACT: Royal College of Surgeon (RCS) rats undergo retinal degeneration due to the inability of retinal pigment epithelial (RPE) cells to phagocytose shed outer segments. We explored the effect of introducing Schwann cells to the subretinal space of RCS rats (before the onset of retinal degeneration), by relying on electroretinogram (ERG) recordings and correlative retinal morphology. Scotopic ERGs recorded from cell-injected eyes showed preserved amplitudes of mixed a-wave b-wave, rod b-waves, and cone b-waves over controls (sham-injected eyes); photopic b-wave amplitudes and critical flicker fusion were also improved. Normal retinal morphology was found in areas of retinas that had received cell injections. Since Schwann cells have no phagocytic properties, their therapeutic effect is best explained through a paracrine mechanism (secretion of factors that ensure photoreceptor survival).
    Vision research 07/2009; 49(16):2067-77. DOI:10.1016/j.visres.2009.05.014 · 1.82 Impact Factor
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    • "The RCS rat is used widely to study retinal degeneration and to develop experimental therapies directed at limiting the progress of photoreceptor loss (Eisenfeld et al., 1984; Sheedlo et al., 1989; Li & Turner, 1991; Lin et al., 1996; Little et al., 1996; LaVail, 2001; Lund et al., 2001, 2003; Vollrath et al., 2001). Most anatomical studies have focused on thickness of the outer nuclear layer as the index of the progress of degeneration and the effects of treatment. "
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    ABSTRACT: The Royal College of Surgeons (RCS) rat has a retinal pigment epithelial cell defect that causes progressive loss of photoreceptors. Although it is extensively used in retinal degeneration and repair studies, how photoreceptor degeneration affects retinal circuitry has not been fully explored. This study examined the changes in synaptic connectivity between photoreceptors and their target cells using immunocytochemistry and correlated these changes with retinal function using the electroretinogram (ERG). Immunostaining with bassoon and synaptophysin (as presynaptic markers) and metabotropic glutamate receptor (mGluR6, a postsynaptic marker for ON-bipolar dendrites) was already impaired at postnatal day (P) 21 and progressively lost with infrequent pairing of presynaptic and postsynaptic elements at P60. By P90 to P120, staining became increasingly patchy and was eventually restricted to sparsely and irregularly distributed foci in which the normal pairing of presynaptic and postsynaptic markers was lost. ERG results showed that mixed scotopic a-waves and b-waves were already reduced by P21 but not oscillatory potentials. While cone-driven responses (photopic b-wave) reached normal levels at P30, they were impaired by P60 but could still be recorded at P120, although with reduced amplitude; rod responses never reached normal amplitudes. Thus, only cone-driven activity attained normal levels, but declined rapidly thereafter. In conclusion, the synaptic markers associated with photoreceptors and processes of bipolar and horizontal cells show abnormalities prior to significant photoreceptor loss. These changes are paralleled with the deterioration of specific aspects of ERG responsiveness with age. Besides providing information on the effects of photoreceptor dysfunction and loss on connection patterns in the retina, the work addresses the more general issue of how disorder of input neurons affects downstream circuitry.
    European Journal of Neuroscience 10/2005; 22(5):1057-72. DOI:10.1111/j.1460-9568.2005.04300.x · 3.18 Impact Factor
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