Morphological and Functional Rescue in RCS Rats after RPE Cell Line Transplantation at a Later Stage of Degeneration

Casey Eye Institute, Oregon Health Sciences University, Portland, OR 97239, USA.
Investigative Ophthalmology &amp Visual Science (Impact Factor: 3.4). 02/2008; 49(1):416-21. DOI: 10.1167/iovs.07-0992
Source: PubMed


It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19.
Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression.
Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors.
RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

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Available from: Shaomei Wang, Oct 05, 2015
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    • "retinal degeneration accompanying inner retinal changes and neovascularization (Villegas-Perez et al., 1998, Wang et al., 2003). We have shown that subretinal injection of RPE cells, Schwann cells, mesenchymal stem cells and neural progenitors at an early stage of the disease can substantially slow down vision loss (Lawrence et al., 2004; Wang et al., 2005b; Gamm et al., 2007; Wang et al., 2008; Lu et al., 2009; Lu et al., 2010). In addition, the secondary pathological changes are also largely limited in the graft-protected retinal area. "
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    ABSTRACT: The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.
    Visual Neuroscience 03/2013; 30(3):1-13. DOI:10.1017/S0952523813000011 · 2.21 Impact Factor
    • "Stem cell therapy is a potentially effective strategy in helping to treat retinitis pigmentosa (RP) and age-related macular degeneration (AMD). To date, researchers have studied different sources of donor cells such as mature photoreceptors [21], retinal progenitor cells (RPC) [26], retinal sheets [15] and RPE [28] for cell transplantation to the subretinal space, and have confirmed varying degrees of enhanced vision in animal models [15] [18] [20] [28]. However , the clinical use of these cells has presented problems including complications of surgical procedures for extraction [10], restricted accessibility of pluripotent retinal stem cells, and the formation of cell orientation in rosettes [2] [19]. "
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    ABSTRACT: Stem cell therapy is a promising approach for treatment of degenerative retinal disorders such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In this study, human mesenchymal stem cells (MSCs) were isolated from the trabecular meshwork (TM), the major functional tissue of the anterior chamber angle in the eye, were characterized and differentiated into photoreceptor cells on amniotic membrane (AM). After isolation of trabecular meshwork and culture of the stromal segment of this tissue, fibroblast-like cells (CD105(+), CD90(+), CD44(+), CD166(+) cells) capable of differentiation toward mesenchymal and photoreceptor lineages were obtained. The isolated cells were seeded on amniotic membrane and were treated with induction medium. Immunocytochemistry and quantitative real time RT-PCR (qPCR) were used to detect expression of photoreceptor genes such as rhodopsin, recoverin, CRX, and peripherin; and the bipolar cell marker protein kinase C alpha (PKC-alpha). As a result, immunocytochemistry revealed that the differentiated TMMSCs expressed rhodopsin, CRX and PKC proteins. qPCR showed the expression of rhodopsin (rod like photoreceptor-specific marker), and CRX genes were significantly higher in TMMSCs differentiated on AM than those differentiated on tissue culture polystyrene (TCPS). In conclusion, our findings suggested that a combination of TMMSCs (as a new source) and basement membrane support from AM might be a suitable source of cells for subretinal transplantation in regenerative therapy for retinal disorders such as AMD and RP.
    Neuroscience Letters 02/2013; 541. DOI:10.1016/j.neulet.2012.12.055 · 2.03 Impact Factor
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    • "Several groups have used different cell lines as a source for retinal transplant, as the E1A cell line (Seigel et al., 1998), ARPE-19 (Pinilla et al., 2007; Wang et al., 2008), non-retinal cells (Pinilla et al., 2009) or neural progenitor cells (Gamm et al., 2007). Giannelli et al. (2011) differentiated human Müller cells to rod photoreceptors with an efficiency of 54% and showed that Müller-derived photoreceptors integrate and survive within immunodeficient mouse retinas (Giannelli et al., 2011). "
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    ABSTRACT: Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT(1) and MT(2) receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.
    Experimental Eye Research 01/2013; 110. DOI:10.1016/j.exer.2012.12.006 · 2.71 Impact Factor
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