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Producing eye cellsRPE and Neural Retina from human embryonic and adult iPS type stem cells
Past and present work
In the absence of appropriate intercellular signals specifying alternate cellular fates, embryonic stem cells (ESCs) may be directed towards neuronal differentiation by autocrine signals (Hemmati-Brivanlou & Melton, 1997; Tropepe et al., 2001; Munoz-Sanjuan & Brivanlou, 2002; Lowell et al., 2006). The apparent ease by which neural progenitor cells have been derived from cultures of Human Embryonic Stem Cells (HESCs) may partly explain the ability of these cells to differentiate towards neuroretinal lineages including RPE and photoreceptor precursors.
Differentiation of RPE cells from ESCs
Although melanocytes have been known to form from mouse ESCs for almost a decade (Yamane et al., 1999), it is only more recently that pigmented cells derived from ESC lines have been identified as RPE cell-like (Kawasaki et al., 2002; Hirano et al., 2003; Ooto et al., 2003).
Kawasaki and colleagues were the first to use a cell culture method employing stromal cell-derived inducing activity (SDIA) from PA6 stromal cells to derive pigmented, Pax6 positive cells from primate ESC cultures (Kawasaki et al., 2002). Two subsequent studies confirmed the ability of SDIA to induce RPE cells from mouse and primate ESCs (Hirano et al., 2003; Ooto et al., 2003).
More recently, it has been shown that Wnt2b signalling can increase the production of RPE cells from SDIA-cultured mouse ESCs (Aoki et al., 2006). The most recent modification to the SDIA induction protocol involves dispensing with these cells entirely and using instead the matrix layers of human amniotic membrane (Ueno et al., 2006). This may prove to be an important innovation for future derivation of clinically applicable RPE cells. At present, the exact molecular nature of SDIA and amniotic membrane matrix signals are unknown.
The molecular RPE phenotype of pigmented HESC-derived cells has been confirmed by a comprehensive study (Klimanskaya et al., 2004). Here, a comparative transcriptomics approach was adopted to assess the similarities (in terms of gene expression profile) between HESC-derived RPE cells, human foetal RPE and several human RPE cell lines. Rather encouragingly, HESC-derived RPE cells not only produce mRNA specific to RPE cells (e.g. RPE65) but they actually appear more similar to human foetal RPE (in terms of transcriptional profile) than the other human RPE cell lines tested. In addition to transcriptional profiling, the study by Klimanskaya and colleagues (2004) also provides evidence that HESC-derived RPE cells can phagocytose latex beads and express two RPE-associated genes at the protein level: CRALBP and bestrophin.
The reports from Robert Lanza and colleagues, together with our own observations (Vugleret al., 2007) of RPE derivation from Sheffield HESCs suggest that for many HESC lines, derivation of RPE cells may be a spontaneous phenomenon under conditions of co-culture with mouse embryonic fibroblasts.