20.5Challenges for RPE Stem Cell Therapy

The use of stem cells for RPE repair represents an exciting possibility. Each stem cell population has its advantages. Embryonic stem cells have considerable plasticity and have been shown to be totipotent, differentiating to all lineages. ES cells are limitless in their numbers and thus would represent an endless supply of cells for therapeutic use. The risk, however small, still remains that they may undergo possible malignant transformation.

The ability of adult stem cells in a specific organ to generate cells of unrelated types decreases in the more committed progenitors. However, mounting evidence suggests that the initial differentiation into one specific cell type is not as irreversible as originally thought [50, 51] and recent findings, especially in bone marrow stromal cells (BMSCs), suggest that the lineage commitment of a stem/progenitor cells is not absolute [52].

The use of autologous cells still remains the best option, as there will not be any need for immune suppression or risk of rejection. Yet this approach has limitations including that often the tissue needing repair cannot be a source of reparative cells. Thus, the approach we favor is the enhanced differentiation of endogenous BMDCs, or a particular bone marrow cell hematopoietic population, to an RPE-like phenotype. We specifically utilized targeted gene manipulation to promote differentiation in adult stem cells. We hypothesized that expressing a gene unique to a terminally differentiated cell type, and with secondary effects on transcriptional modulation, could promote BMSC differentiation more readily into the obligatory cell type, thus enhancing the repair process [1].

One candidate for directing BMSC differentiation into RPE is the RPE-specific protein RPE65. RPE65 is critical for the normal formation of 11-cis retinal and thus photoreceptor function. RPE65 modulates the availability of retinoic acid, a known transcriptional regulator and differentiation inducer [53–60]. Furthermore, RPE65 may “moonlight” as a transcriptional regulator or have other novel functions that enable it to regulate differentiation. We showed that genetic manipulation of BMDCs to express RPE65 promotes neuroepithelial cell differentiation, retinal repair and, most importantly, recovery of visual function [1] (Fig. 20.1). These observations provide the first demonstration that adult stem cells can be programmed down a particular differentiation pathway by expression of a differentiation protein that dictates cell specificity.

Fig. 20.1 (continued) and apparent rescue of RPE by RPE65-transfected BMDC. (a) An eye that was injected with 100 mg/kg sodium iodate but was not given any rescuing BMDC. Note the complete absence of the photoreceptor layer and near absence of any RPE cells. (b) Animals receiving RPE65-infected BMDC have abundant pigmented RPE-like cells on Bruch’s membrane by 28 days posttreatment. (c) Immunohistochemical localization of GFP+ cells coexpressing the RPEspecific marker CRALBP to the correct anatomical locale in the sodium iodate-injured eye confirming both their BMDC origin and RPE phenotype

This approach would allow the use of an individual’s own cells. If we could enhance the BMDC to become the cell type in need of repair, then this would represent a viable therapeutic approach. With BMDCs, this process requires not only precise differentiation into RPE but also sufficient BMDC recruitment to and proliferation at the site of injury to restore proper cellular function. An added benefit of using BMDCs is that they are blood borne cells that can be carried by the circulation to the tissue in need of repair; thus, with regard to RPE repair, there would be no need for damaging intraocular or subretinal delivery as other stem cell approaches currently require. BMDCs can be easily removed and readministered after pharmacological or gene manipulation, thus allowing for autologous transplantation. Furthermore, adoptive transfer of these stem cells is minimally invasive. Adult stem cells also have an advantage over embryonic stem cells which, despite their robust ability to proliferate as well as differentiate, have at times resulted in unfavorable outcomes such as development of teratomas and neoplasia [61].

External signals in the stem cell microenvironment (cytokines and matrix) provide cues to control cell fate decision in terms of proliferation or differentiation into a desired, specific phenotype. Stem cells respond to both temporal and spatial signals.

Developmental studies suggest that a number of critical genes regulate embryonic cell differentiation, e.g., Pax-6, Nanog, and Olig1, and targeted gene manipulation of embryonic stem cells with specific transcription factors have promoted cellspecific differentiation [62–64]. However, the drivers for adult stem cell differentiation are more elusive. For BMDCs, VEGF and SDF-1 are critical regulators of their differentiation into the endothelial cell linage. Retinoic acid regulates limbal stem cell differentiation into corneal epithelial cells [65]. Hepatocyte growth factor (HGF) promotes the differentiation of BMSC-derived oval cells into hepatocytes in vivo [66]. Furthermore, the recognition that gene products such as growth factors can act as transcriptional regulators, either directly or via metabolites, implies that proteins unique to a cell phenotype may play a critical role in the terminal differentiation to that particular cell type.

Tissue injury with its resultant loss of cellular function and loss of tissue architecture recapitulates aspects of development. Tissue repair reestablishes cellular order and functional specialization much like cellular differentiation does in development. Both processes, tissue repair and development, utilize immature undifferentiated cells that succumb to the influence of transcriptional activators in a specific temporal pattern [67]. Gene modulation of adult stem cells thus may provide an efficient source of RPE cells that can be used in treatment of RPE diseases. Furthermore, the same paradigm, well established in development, may allow the preprogramming of undifferentiated adult stem cells to express cell type specific genes and ultimately become that specific cell type. Turning on transcriptional activators at specific times by cell type specific factors may allow BMDCs to be used as a cell therapy for a wide variety of diseased tissues.

20.6 Characterization of RPE-Like Cells Derived from BMDCs

Over a decade ago, Limb et al. determined that hematopoietic cell markers, including all isoforms of CD45, were constitutively expressed on RPE cells, that expression of hematopoietic molecules by RPE cells may influence the macrophage-like properties of these cells and may also aid in the identification of RPE cells during pathological processes, particularly in the proliferative retinopathies, where these cells undergo phenotypic and functional changes [68]. As stated above, more direct evidence that RPE cells have a link with the hematopoietic system is the findings of several groups that have demonstrated that BMDCs can give rise to RPE-like cells.

BMDCs can home to, and regenerate the RPE after induced injury [34]. For these studies, two types of injury were performed: physical damage of Bruch’s membrane with a needle in GFP chimeric mice or RPE damage by sodium iodate injection into albino mice (tyrosinase gene knockout mice) undergoing transplant with cKit+ BMDCs from pigmented mice (mice with normal tyrosinase gene). Injury to the RPE recruits BMDCs to incorporate into the RPE layer and differentiate into an RPE phenotype. In this study, a portion of the BMDCs adopted RPE morphology, expressed melanosomes, and integrated into the RPE without cell fusion [34]. It was concluded that BMDCs can migrate to the RPE layer after physical or chemical injury and regenerate a portion of the damaged cell layer.

The importance of the CD133+ cell population within the cKit+ enriched hematopoietic compartment has been documented [69] for intravitreal injections of CD133+ hematopoietic progenitor cells improves visual function. CD133 was chosen because it is an enrichment marker for multipotent hematopoietic progenitor cells [70]. It is also expressed on a variety of tissue specific stem/progenitor cells, and functional loss of CD133 or prominin-1 leads to retinal degeneration in humans [71]. The CD133 transplanted cell population homed to the damaged RPE, largely by CXCL12 signaling, assumed RPE morphology, expressed pigment, and expressed the RPE specific genes RPE65 and CRALBP. In addition, CD133+ cells, and not CD133- cells, provided functional protection of the photoreceptor electroretinogram (ERG) b-wave 18 days after sodium iodate injury. Importantly, human CD133+ cord blood cells also regenerated pigmented RPE cells in a xenograft model [69]. These animal experiments show that CD133+ progenitor cells with a myeloid phenotype migrate to damaged RPE and assume RPE-like morphology and function. This is not surprising considering myeloid cells home to sites of tissue damage and are known to be pleiotropic.

Li et al., investigated whether bone marrow-derived cells (BMDCs) can be induced to express RPE cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo [72]. Adult RPE cells were cocultured with GFP-labeled stem cell antigen-1 positive (Sca-1+) BMDCs for 1, 2, and 3 weeks. BMDCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell–cell contact with RPE. Using an animal

model of sodium iodate-induced RPE degeneration, BMDCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. BMDCs were identified in the subretinal space as Sca-1+ or c-kit+ cells and they were double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on Bruch’s membrane in focal areas of RPE damage. These authors concluded that BMDCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage [72].

Atmaca-Sonmez et al. also used GFP-labeled cells of bone marrow origin in a sodium iodate model of RPE damage in the mouse [73]. At both 1 and 2 weeks after intravenous injection, GFP+ cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of BMDCs plus facilitating T cells (FC) appeared to favor the survival of the homed stem cells at 2 weeks and the RPE-specific marker RPE65 was expressed by adoptively transferred BMDCs by 4 weeks. They concluded that systemically injected BMDCs homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, RPE65 was expressed on adoptively transferred BMDCs in the denuded areas [73].

Transducing BMSC with an adenovirus facilitated their differentiation into RPE-like cells [33]. An adenoviral vector expressing either GFP or pigment epithelialderived factor (PEDF) was use to transduce rat BMSCs in vitro before subretinal transplantation into either control rats or RCS rats. Two months after cell injection, some adenovirus-PEDF treated rat BMSCs integrated into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells expressed the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects were observed following grafting of these vector-transduced and nontransduced BMSCs in semi-thin sections of dystrophic retinas. Ultrastructurally, BMSCs were detected on top of host RPE and in close contact with photoreceptor outer segments and were found to be phagocytosing rod outer segments, raising the possibility that BMSCs have the potential to replace diseased RPE cells if delivered into the subretinal space, and may protect photoreceptor cells from degeneration [33].

Li et al. characterized chemoattractants expressed by the RPE after sodium iodate-induced damage and investigated whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE [74]. mRNA for SDF-1, C3, HGF, and leukemia inhibitory factor (LIF) was significantly increased and higher SDF-1 and C3 protein secretion from the RPE was found after sodium iodate treatment. Increased expression of early ocular markers in peripheral blood mononuclear cells was observed after mobilization. The conclusion of these studies was that damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells. They also concluded that retina-committed stem cells appear to reside in the BM and can be mobilized into the peripheral blood by granulocyte colony stimulatory factor and flt3-ligand and that these stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage [74]. However, this interesting observation that RPE-committed stem cells reside in the bone marrow remains to be confirmed by another laboratory.