Celia F. Nunes, Ilene K. Sugino, Jianqiu Wang, Aprille Rapista, Christopher Malcuit, Noounanong Cheewatrakoolpong, Adam C. Johnson, Irina Klimanskaya, Qian Sun, Robert Lanza, and Marco A. Zarbin
Cell-based therapy involving RPE transplantation might preserve or restore vision in AMD patients with evolving atrophy or in patients with other diseases in which vision loss is associated with dysfunctional RPE. Cell transplantation in patients with AMD has been attempted using a number of cell types and preparations, including fetal and adult RPE (autologous and allogeneic), translocated autologous choroid/RPE, and autologous iris pigment epithelium (IPE; see review by Binder1). Transplantation of autologous RPE and IPE is attractive because there is no risk of immune rejection. However, older cells: (1) do not behave as robustly as those from young donors,2–4 (2) may carry AMD-related gene defects or modifications caused by aging,1,5,6 and (3) may not have the ability to perform all the functions necessary to maintain the photoreceptors.5 Because fetal human RPE begin to show morphologic abnormalities after five to six passages, they are not suitable as a “universal” donor source, regardless of the possible immunogenicity of such cells.7 In addition, the supply of RPE from young donors is limited, so it would not be practical to develop a RPE transplant paradigm based on the use of such cells. Embryonic stem cells offer an advantage over fetal or adult RPE because of their ability to undergo large-scale expansion, assuring an abundant supply of well characterized, pathogen-free cells that can be manufactured in a manner compatible with clinical practice.8 Genetic analysis of such cells shows a high degree of similarity to in situ RPE.9 The method to generate RPE derived from human embryonic stem cells (hES-RPE) is reproducible and can be achieved in a manner that does not cause embryo destruction.10 Manipulation of hES-RPE in culture could take advantage of stem cell plasticity to optimize their ability to attach and survive on aged or diseased Bruch's membrane (BM) and to minimize rejection.11 To assess the potential of hES-RPE for cell replacement therapy in AMD patients, we compared the attachment and survival of hES-RPE of different degrees of pigmentation on BM with cultured human fetal RPE (fRPE) whose behavior has been characterized previously on aged and AMD BM.4,12,13 The goals of this study were to determine: (1) whether hES-RPE have the potential to attach and survive on aged BM; (2) whether a characteristic integrin mRNA profile can predict attachment and/or survival; (3) whether hES-RPE and fRPE have similar morphology after attachment to and growth on BM; and (4) whether hES-RPE secrete neurotrophic proteins after attachment and survival on aged human BM. Using the same hES-RPE preparations as in the present study, Lu et al.8 demonstrated long-term safety and functionality of hES-RPE after subretinal injection in rodents. These and other studies using hES-RPE derived in a similar fashion from spontaneously forming pigmented colonies in confluent hES cultures have shown that hES-RPE express RPE-specific genes, phagocytose outer segments, show polarization of Na+/K+ ATPase, and exhibit morphologic features of RPE.8,9,14–16 Therefore, hES-RPE might serve well for RPE replacement therapy in patients with retinal degenerations where the primary cause of vision loss is diseased or missing RPE. Although animal studies show that hES-RPE can survive in the subretinal space and rescue photoreceptors,8,14,16,17 such studies do not always predict the ability of cells to survive on diseased BM in AMD patients.18 Integrins are important for RPE attachment to BM (reviewed by Afshari19), and alpha integrin subunits have an important role in RPE adhesion to this surface.20 In cultured cells, integrin expression can be modulated by the degree of and time at confluence,21 and integrin upregulation through culturing improves RPE and iris pigment epithelium (IPE) adhesion to BM.4,20,22 One criterion by which one might assess hES-RPE as candidates for transplantation in AMD eyes is the presence of an integrin mRNA profile compatible with attachment to BM (e.g., a profile similar to that of cultured fRPE), which show robust attachment to BM by 24 hours after seeding.4,12,20 To determine whether the integrin mRNA profile can predict successful hES-RPE adhesion and survival on aged submacular human BM, we compared integrin mRNA expression and survival on BM of three different batches of hES-RPE of different degrees of pigmentation (harvested at increasing times in culture) with that of fRPE. In addition to replacing lost or diseased RPE cells with cells capable of performing RPE functions, transplanted cells could rescue dying photoreceptors through the secretion of proteins such as neurotrophic factors and cytokines. This idea is supported by in vivo studies showing that non-RPE cells can rescue photoreceptors despite their inability to phagocytose outer segments.23–26 Therefore, even if the cells do not appear to be fully differentiated on BM after transplantation, secretion of neuroprotective growth factors might have a rescue effect on the overlying retina. As a result, in the studies reported here, we also compared growth factor and cytokine secretion into the media overlying BM explants on which hES-RPE and fRPE were seeded. more...