Article

Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells.

Department of Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
Visual Neuroscience (Impact Factor: 1.48). 03/2012; 29(2):83-93. DOI: 10.1017/S0952523812000041
Source: PubMed

ABSTRACT Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch's membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host's cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

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    ABSTRACT: PURPOSE. TO EVALUATE CELL SURVIVAL AND TUMORIGENICITY OF HUMAN EMBRYONIC STEM CELL-DERIVED RETINAL PIGMENT EPITHELIUM (HESC-RPE) TRANSPLANTATION IN IMMUNOCOMPROMISED NUDE RATS; CELLS WERE TRANSPLANTED AS A CELL SUSPENSION (CS) OR AS A POLARIZED MONOLAYER PLATED ON A PARYLENE MEMBRANE (PM). METHODS. SIXTY-NINE RATS (38 MALE: 31 female) were surgically implanted with CS (n=33) or PM (n=36). Cohort subsets were sacrificed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage) and anti-cytokeratin (epithelial marker). RESULTS. The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P=0.006) and the presence of a monolayer (P<0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. CONCLUSION. hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.
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