AMD is a condition associated with the progressive dysfunction of RPE cells, leading to the death of photoreceptors and eventual central vision loss. The RPE is a monolayer of cells, which underlies the retina, and as such, it offers an accessible target site for treatment of AMD. Proof of concept for the use of human cells in grafting to preserve visual function has been demonstrated in studies involving transplantation of various cell types into the subretinal space of dystrophic animals [31
], including RPE [3
], and in human RPE transplantation trials [35
]. The pluripotent potential of embryonic stem cells makes them an ideal source of material for use in regenerative medicine and cellular therapy. Several papers have described the potential of HESC to differentiate into RPE cells in vitro [1
]. In culture, stem cells differentiate to form monolayers of pigmented cobblestone-like cells that express markers specific to RPE cells including bestrophin, CRALBP, and RPE65 [1
]. Cells are also polarized, with microvilli observed in the apical portion of the cell and the formation of a basement membrane. Although functional analysis of HESC-derived RPE demonstrates that they are capable of phagocytosing latex beads [1
], in vivo, RPE cells will normally phagocytose only POS. Despite the characterization of HESC-derived RPE, previous studies have not addressed the phagocytosis of POS.
The phagocytosis of POS is crucial for the survival of photoreceptor cells; dysfunction in this process leads to clinical disorders characterized by the degeneration of the retina and eventual blindness. Here, using confocal microscopy, we showed that monolayers of HESC-derived RPE cells are able to bind and internalize fluorescently labeled porcine POS and outer segments from a pig retina explant, as indicated by the incorporation of rhodopsin-positive POS fragments within the cells. Over time there was a continual increase in the number of fluorescently labeled POS associated with HESC-derived RPE cells. No saturation of ingestion was observed over the 20 h period of exposure, which is in agreement with studies of phagocytosis in primary human RPE cell cultures [37
Of specific interest, when assessing cells for use in human clinical retinal therapies, is their ability to phagocytose human POS. Due to the limited supply of viable retina tissue, we designed a novel assay, whereby fresh human retina explants were co-cultured with HESC-derived RPE cells in an ex vivo model system. Using electron microscopy, we showed phagocytosis of POS from human retinal tissue by the HESC-derived RPE. We have been able to observe various stages of phagocytosis including the interaction of the POS with apical microvilli and their engulfment into the cell. After 48 h in co-culture with the retina, numerous lipid inclusions were observed within the basal portions of cells, indicative of terminal stages of segment digestion. In future, it will be important to examine phagocytosis of POS by RPE cells after transplantation into animal models of retinal dystrophy to demonstrate that photoreceptor survival is due to RPE-specific functions by donor cells rather than to growth factor-mediated rescue.
Phagocytosis of POS by RPE cells is a complex process and as yet, the full mechanisms responsible remain unclear. Several genes implicated in the three stages of RPE phagocytosis (recognition, ingestion, and digestion) are expressed in HESC-derived RPE and control cells, including αv integrin
and β5 integrin
, cathepsin D
, the clathrin
heavy and light chains, focal adhesion kinase
, and ProS
. We have also confirmed the expression of αVβ5, MERTK, and FAK proteins by the cells using western blot and immunocytochemistry. Control HESC also express this range of molecules which indicates that, once confluent, non-pigmented precursors expressing RPE cell markers are present within the population of cells before the removal of bFGF. These precursors can be identified using the antibody Pmel17, which stains pre-melanosomes [4
]. Of particular interest to us is the expression of full-length (180 kDa) MERTK in HESC-derived RPE cells, which we have demonstrated to be crucial to the specific phagocytosis of outer segments. Using an antibody against MERTK to block function inhibits the uptake of outer segments, but has no effect on the ingestion of latex beads, suggesting that the specific phagocytosis of outer segments by HESC-RPE cells is mediated through the MERTK receptor, while nonspecific phagocytosis (i.e., polystyrene beads) is mediated through a separate mechanism.
It is worth noting that of all the molecules examined, MERTK was the only one not detected in our stock of ARPE-19 cells at the mRNA or protein level under the described culture conditions. Mutations in MERTK, which underlie the dystrophic phenotype observed in the RCS rat [22
] and some human forms of retinitis pigmentosa [23
], revealed the importance of this protein in the maintenance of a healthy retina. It is known that the gene expression profile of HESC-derived RPE more closely resembles that of freshly isolated RPE, when compared with human RPE cell lines [1
]. Accordingly, this also appears to be true at the protein level. Even though ARPE-19 cell transplantation has beneficial effects in rats [34
], the complete absence of MERTK and other molecules critical to RPE cell function [1
] suggest that caution should be taken when investigating established RPE cell lines for use in human transplantation therapies. However ARPE-19 cells may prove to be a useful model system for unraveling the effects of the absence of MERTK in a human cell line.
The morphological appearance of HESC-derived RPE cells resembles that of the RPE in vivo before co-culture. However it is interesting to note that, after 48 h co-culture with human retina, cells appeared more mature. Structural changes observed in the cells are reminiscent of those observed in the rat RPE postpartum, with increases in the length of apical microvilli and the appearance of basal membrane infolding [5
]. We observed an increase in the number of melanosomes, and in particular the number of late stage 3–4 melanosomes within the apical region of the cells. These findings concur with studies in vivo, suggesting that injections of ROS can induce melanogenesis in adult rat RPE cells over 5 days [42
]. The shorter time period of melanogenesis observed in these cells may well reflect the immature state of HESC-derived RPE, having never been exposed to a POS. Other changes within the cells suggests that exposure to POS/retina may contribute to the functional maturity of the RPE. The presence of coated pits, and the emergence of coated vesicles and basement membrane infoldings are all signs of increased functional potential, suggesting the HESC-derived RPE are regulating cellular transport via the endocytosis of substances at the apical surface and the exocytosis of substances at the basal membrane. Tight junctions regulate the diffusion of substances across epithelial cell monolayers and function in the RPE as an integral component of the blood:retina barrier. The presence of tight junctions and their associated proteins [4
] in HESC-derived RPE provides further evidence to suggest that these cells possess critical barrier properties.
One of the inherent problems of cell culture is the removal of cells from their original tissue source. Although adult RPE cells grow well in culture, repeated passage of cells derived from human RPE tissue results in the differentiation of cells away from their original phenotype, typified by high levels of proliferation, changes in gene and protein expression, decreased levels of pigmentation, and loss of structural characteristics [4
]. Together these data demonstrate how critical the microenvironment is in the maintenance/restoration of RPE cell lineage. Previous studies have demonstrated that manipulation of the microenvironment has profound effects on RPE cells, specifically, the introduction of POS can induce changes in gene expression [44
], tyrosinase biosynthesis and activity [47
], and, pertinent to data presented here, increased levels of melanogenesis [42
]. It is clear that RPE cells can differentiate from HESC in culture [1
]; however, full structural and functional maturation may require in vivo signals from the retina. These findings also have implications for the differentiation of stem cells for clinical therapies. Perhaps only when cells are transplanted into the appropriate environment will full differentiation and functional maturation be observed.
In conclusion, we have demonstrated that HESC-derived RPE have the necessary molecular profile for the specific phagocytosis of outer segments, and require MERTK to mediate this process. Using a novel in vitro system, we have shown that HESC-derived RPE cells can phagocytose human outer segments, since there is evidence of engulfment and end stage lipid accumulations. Additionally, co-culture with retinal tissue may contribute to structural changes in RPE cells associated with maturation and increased functionality.