Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.

Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics.

Pigmentation is a typical feature for retinal pigment epithelial cells (RPE), and each RPE cell represents individual characteristics. Although, type and intensity of pigmentation may vary, still most cells are pigmented to some extent. In this study, we setup a standardized quantitative melanin content analysis for human embryonic stem cell (hESC) derived RPEs (hESC-RPE) and compared this method to an image based pigment quantification technique. The optical quantification of pigmentation was done from micrographs of hESC-RPE. The extracted pigment was quantitated with spectral analysis, with UV-Vis spectroscopy, and the size-distribution with dynamic light scattering. The results revealed that the measured particle sizes of extracted melanin corresponded to known sizes extracted melanosomes. In addition, the optical pigmentation and absolute melanin concentration were clearly correlating. Our data suggests that image analysis and quantitative melanin content analysis can be interchangeably utilized in pigmentation quantitation: if the relative pigmentation of hESC-RPE cells is desired to be estimated with a non-invasive method then image analysis is the choice of method, but if the exact amount of melanin needs to be evaluated, then the new melanin extraction method presented here, should be chosen.

Summary Human embryonic stem cell‐derived retinal pigment epithelial cells (hESC‐RPE) provide a promising cell source for studying retinal development, for disease modelling, and retinal cell replacement therapies. Several research groups, including ours, have demonstrated that hESC‐RPE structure, function, and physiology resembles that of native RPE regarding cellular fine structure and expression of many RPE signature genes and proteins. To characterize the hESC‐RPE proteome in larger scale, we have compared hESC‐RPE protein expression to primary human RPE using isobaric tags for relative quantitation (iTRAQ) technology. The hESC‐RPE proteome reflected that of native RPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No adverse signs such as increased stress, proliferation, or retinal degeneration‐related changes were seen in hESC‐RPE, while proteins involved in key RPE functions such as visual cycle and phagocytosis were detected. Proteomics provides valuable tools for validation of hESC‐RPE, studying disease mechanisms, and discovery of new biomarkers and therapeutic targets.