The RPE is critical for retinal viability and function and has been the focus for AMD and its therapeutic interventions. Although a significant amount of research has focussed on deriving functional RPE cells from various stem cell sources, little is known about the global transcriptional profiles of these stem-cell-derived RPE cells. In this study, we have derived functional RPE cells from various lines of hESCs and hiPSCs and generated global expression profiles of stem-cell-derived RPE and human fRPE cells. We found that hESC-RPE resembles fRPE more closely than hiPSC-RPE. The expression profiles of hiPSC-RPE suggest that they are in a relatively immature differentiation state. Also, we identified a set of genes that are exclusively expressed in human fRPE. This set of genes may serve as reliable molecular signatures of fRPE and provide future standards for scoring the differentiation state of stem-cell-derived RPE cells. Together, these results offer critical insights into the therapeutic use of stem-cell-derived RPE cells in treating AMD.
Stem-cell-derived RPE cells have a highly similar morphology to human fRPE. These cells arise spontaneously from stem cell sources 3–4 weeks after differentiation, consistent with the differentiation timeline shown by other groups. We observed varying differentiation propensities between cell lines. This may be a result of genetic background differences between each cell line, especially in hiPSCs. Previous reports have shown some cell types are more efficiently reprogrammed (7
); it is possible that these biases are persistent during differentiation to RPE cells.
HESC-RPE and hiPSC-RPE express a panel of RPE gene transcripts similar to cultured fRPE cells. Co-expression of the early RPE development marker, PAX6, with various mature markers suggests that hESC-RPE has some heterogeneity, which may be due to immature cells around the leading edges of hESC-RPE expanding sheets. HESC-RPE were previously shown to expand through a de-differentiation mechanism, which involves pigmented cells showing de-pigmentation, then entering into the active cell cycle before gaining pigmentation again (12
). Interestingly, a majority of the RPE-associated markers was detected in hiPSC-RPE, but not the early marker, PAX6, and the late marker, LRAT, suggesting that hiPSC-RPE may be in a unique differentiation state. Recent reports have shown some loci are more resistant to reprogramming in hiPSCs, which may affect the expression of certain genes (9
). There appear to be several discrepancies between hESC-RPE, hiPSC-RPE and primary RPE cells, but it remains unclear whether these differences will affect the utility of stem-cell-derived RPE in the clinical setting.
Global gene expression analysis demonstrated that hESC-RPE resembles fRPE more closely than hiPSC-RPE. Pair-wise comparisons between hESC-RPE to fRPE and hiPSC-RPE to fRPE revealed that stem-cell-derived RPE cells show under-expression of genes involved in visual perception. Interestingly, some of the up-regulated transcripts in hiPSC-RPE are involved in immune responses. This observation is consistent with transcriptional and methylation profile comparisons between hESCs and hiPSCs (unpublished data), suggesting hiPSC-RPE may retain specific reprogramming gene signatures.
Furthermore, we found similar gene expression levels of complement associated proteins previously reported to be associated with AMD in stem-cell-derived RPE and fRPE cells. Allele variants for different complement cascade-associated proteins have been implicated as major risk factors for the predisposition of AMD (25
). Further analysis of SNPs in each stem cell line is required to determine whether they carry AMD risk variants. Our analysis of aging retina-associated genes shows that cultured stem-cell RPE mimic fRPE and do not show signs of aging. This addresses the concern that extended culture may induce the expression of age-related genes and suggests that culture-derived RPE may be suitable replacements for fRPEs in cell transplantation therapy.
Recent microarray-based studies have identified RPE-specific genes by comparing human RPE with various retinal and somatic tissues (20
). However, comparisons between two separate studies showed very few RPE signature genes actually overlapped. These discrepancies may arise from differences in the experimental design and tissues used in their respective analyses. We chose to cross-reference our data with Miller's data set due to the more comprehensive nature of their study (20
). Using their identified signature gene list, we compared their expression with hESCs and refined the list by excluding genes that are not significantly differentially expressed with hESCs. Surprisingly, a small group of eye developmental genes are uniquely expressed in fRPE but not in stem-cell-derived RPE cells, indicating that several eye development pathways remain underdeveloped in stem-cell-derived RPE cells. It is possible that using different differentiation protocols may yield activation of these pathways in stem-cell-derived RPE cells. Previous studies using directed differentiation to recapitulate in vivo
development of RPE first directed differentiation toward the neuronal lineage, followed by differentiation toward the RPE fate. It will be important to examine the differences between spontaneous differentiation and directed differentiation and find optimal culture conditions for deriving RPE cells that best resemble native RPE.
Our results highlight significant differences between expression profiles of stem-cell-derived RPE cells and fRPE. To fully realize the potential of stem-cell-derived RPE in cell-based therapy, future work using in vivo models will elucidate the utility of stem-cell-derived RPE in restoring vision. This study establishes a standard for expanded analyses of expression profiles in additional cell lines. Our findings represent an important step toward optimizing the future application of stem-cell-derived RPE for transplantation into AMD patients.