The successful differentiation of iPS cells into RPE represents a significant advance in the search for a potential cell source for the treatment of human neural retinal diseases. iPS(IMR90)-3 cells readily differentiate into RPE cells and the differentiation protocol used in this paper is a highly efficient method of producing multiple confluent flasks of highly enriched pigmented cells. An efficient initial RPE differentiation protocol is essential for the production of cells for use in any therapeutic application since repeated passaging of RPE results in phenotypical and morphological changes associated with dedifferentiation
. The RPE cells derived from iPS(IMR90)-3 cells have been well-characterized here and satisfy many of the known criteria of RPE cells, including protein expression, cellular pigmentation and polarization. These properties are similar to those observed in cultured HESC-derived RPE
. iPS-RPE also down-regulate the embryonic transcription factors originally used to induce pluripotency from the somatic cell
, suggesting differentiation away from the initial iPS phenotype.
Critical functions of RPE cells include the maintenance of photoreceptor cell integrity by phagocytosing debris shed by the retina each day and epithelial transport of molecules and metabolic waste
. Functional properties of RPE cells are also observed in iPS-RPE in vitro
, including the transport of fluid across cells, as indicated by the formation of blister-like domes in the monolayer
. Similar to HESC-derived RPE, iPS-RPE can phagocytose photoreceptor outer segments from isolated preparations and porcine retina explants
. A recent paper has also described the in vitro
phagocytic properties of iPS-RPE cells
derived using an alternative culture protocol 
, however, the in vivo
function of these cells has yet to be tested.
After 8 days in the sub-retinal space of the dystrophic RCS rat, iPS-RPE cells are capable of phagocytosing host photoreceptor outer segments. This evidence of in vivo phagocytosis is characterised by the presence of an exclusive outer segment marker, rhodopsin, within the cytoplasmic compartment of cells labelled with HSM. These data suggest that human iPS-RPE cells are able to contribute to host photoreceptor cell integrity by removing retinal debris at this time-point.
iPS-RPE cells were transplanted into dystrophic RCS rat eyes at three weeks of age, a time when retinal abnormalities are first observed. The progression of retinal dystrophy in the RCS rat is such that by 13 weeks post-graft most of the ONL has disappeared 
and the photoreceptor outer segment layer is reduced to a debris zone
, a finding we observed in dystrophic controls. The preservation of these layers 13 weeks after iPS-RPE cell injection suggests that the transplantation of these cells preserves retinal structure. At this stage we also performed analysis of visual function. Visual acuity tests showed that the performance of animals receiving iPS-RPE transplants was significantly better than sham-operated and control animals. We also identified the presence of functional neuronal circuitry in the retina after iPS-RPE transplantation as indicated by the light induced c-Fos response in the INL and GCL of the neural retina. This is the first time that that this assay has been used to ascertain retinal function after cellular therapy in a retinal degenerate animal. Importantly, the preservation of light-induced c-Fos expression was restricted to the dorsal retina, surrounding the region of iPS-RPE cell injection only, suggesting preservation of localised neural activation corresponding to the histology of rhodopsin expression. Previous studies, using electroretinography to assess function following cell transplantation into the RCS rat have only been able to demonstrate global activity across the retina 
The absence of iPS-RPE cells in the subretinal space at the time of functional assessment (13 weeks) indicates that the significant benefits observed could not be wholly attributed to the donor cells. Although all animals were maintained on an oral immunosuppression drug, ciclosporin, throughout the experiment, this was not sufficient to sustain iPS-RPE cell survival. These findings are in agreement with previous studies that show that xenografts can be compromised
even after triple immune suppression
. Our analysis suggests that loss of transplanted cells is associated with infiltration of the subretinal space by macrophages/microglia. The large pigmented CD68-positive cells observed in the subretinal space at 13 weeks are likely to be macrophages/microglia filled with melanin
from the transplanted human iPS-RPE cells. The presence of rhodopsin within the macrophages/microglia could explain some of the behavioural and functional benefits observed, since clearance of outer segment debris by these cells in the subretinal space could also contribute to photoreceptor cell survival. This conclusion has been implied previously in a study which suggests that macrophage infiltration in response to the trauma of retinal detachment after saline injection, contributes to extend the longevity of photoreceptor cells in the RCS rat
. Our finding highlights a key aspect of cellular transplantation not fully addressed in many short or long-term studies of RPE transplantation: the host inflammatory/immune response to the xenograft and its indirect role in the preservation of the retina. Importantly, we show that the presence of pigmented cells within the subretinal space does not necessarily reflect survival of transplanted cells. We suggest correct identification of the origin of these cells (using human specific markers, which define cell membranes) is essential in order to distinguish viable donor cells from host inflammatory cells which have engulfed transplanted cells. Identification using pigmentation alone is not sufficient.
The increased visual acuity and outer nuclear layer preservation observed in iPS-RPE injected animals could also be due to a neuroprotective effect produced by the donor cells. RPE are known to secrete neurotrophic growth factors such Glial cell derived neurotrophic factor (GDNF), Brain derived neurotrophic factor (BDNF)
. As these factors can exert protective effects on retinal neurons
and neurons in other neurodegenerative disease models 
, it seems likely that these substances may contribute to the latent photoreceptor cell survival we observe in the dystrophic retina. The fact that the area of preservation extends beyond the borders of the graft suggests that a neuroprotective effect may also be involved. Release of growth factors can occur as a result of surgery but the effects are of shorter duration.
The major limitation of this study is the rejection of human iPS-RPE after transplantation into the RCS rat. RPE cells derived from HESC show long-term viability in vivo
, since in a previous study we have shown that HESC-derived RPE can survive in the RCS subretinal space for up to 10 weeks
, whilst a recent study has indicated survival of cells for up to 30 weeks
. The embryonic origin of HESC-derived RPE may reflect a more immune privileged cell type in comparison to iPS-RPE, which contributes to their longer-term survival after xenografting. HESC have been shown to have reduced immunogenicity, expressing low levels of MHC-I and MHC-II in both the undifferentiated and dedifferentiated state, and to possess an adaptive mechanism to immune responses
. As yet the immunogenic profile of iPS or iPS-derived cells has not been described. Thus, the RCS rat retina offers a useful model for the short-term analysis of iPS-RPE cell function in vivo
, but the donor cell loss due to host macrophage infiltration of the xenograft indicates that additional modification may be necessary to promote long-term donor cell survival in this animal model system.
The major benefit of using iPS cells to treat AMD is that developing a patient specific therapy may help to eliminate the problems associated with immune rejection. Proof of concept for the therapeutic use of a patient's own iPS-derived RPE lies in current clinical treatments for AMD. Although complicated, surgical procedures such as the autologous transplantation of peripheral RPE to the macular region
and macular translocation, where the neural retina is detached and the fovea relocated to a less diseased area of RPE
, have been shown to stabilize visual acuity in AMD patients. Consequently, although iPS-RPE may still carry the same genetic defect responsible for AMD in the patient, the fact that these cells have not been diseased by age, like macular RPE, suggests that they could still be used as a viable therapeutic. iPS cell therapy might also be useful in patients with genetic diseases, such as Leber's congenital amaurosis where transplantation could be combined with gene therapy to correct genetic defects inherent to the patients' own RPE cells. Alternatively iPS cells derived from a tissue-matched healthy sibling may also be useful
. iPS-RPE may also provide a useful in vitro
model system in which to study the pathogenesis of human RPE-linked diseases and identify novel molecular/biochemical therapeutic targets.
While this particular line of iPS-RPE cells could not be used as a direct therapeutic due to viral insertions of pluripotency genes, the recent advances in iPS cell reprogramming technology, including the use of small molecules
, non-integrating episomal vectors
and manipulation of endogenous transcription factors
should eliminate the risks associated with integration of stem cell genes into the genome. Furthermore, the finding that blood cells can be used to derive iPS cells
may remove the need for invasive patient biopsies required for the collection of somatic cells and accelerate the ethical production of stem cell-derived tissue for therapeutic use.