Abstract
Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.
Highlights
The retinal pigment epithelium (RPE) is a polarized monolayer of pigmented cells between the neural retina and choroid
To profile miRNA expression patterns, we constructed small RNA libraries from 1) human embryonic stem cells (ESCs), 2) 15-day partially differentiated human embryonic stem cells (hESCs), 3) early pigmented clusters (PC) that appear around 30 days, and 4) RPE cells in culture for over 3 months [9,18]
Our data indicated that partially differentiated hESCs and immature RPE shared many similar miRNA characteristics, consistent with the notion that miRNA signatures can be used to gauge the state of RPE differentiation. 3D-biplot of the first three principal components further confirmed our hypothesize that HSF1-RPE is immature compared to H9-RPE and hiPS2-RPE, which cluster away from ES and other partially differentiated cells and closer to fully differentiated somatic tissues
Summary
The retinal pigment epithelium (RPE) is a polarized monolayer of pigmented cells between the neural retina and choroid. RPE plays many crucial roles in the eye including blood/retina barrier formation, nutrient transport between blood to photoreceptors, water conveyance from subretinal space to the blood, light absorption, growth factor secretion, and phagocytosis of the outer segment of photoreceptors [1]. For these reasons, dysfunction or death of RPE can lead to photoreceptor degeneration and eventual blindness in diseases such as Stargardt disease and aged related mascular degeneration (AMD) [2,3,4]. The process can take between three to six months to achieve functional stem cell-derived RPE. Other approaches for promoting RPE differentiation treatment with defined factors or small molecules such as niconimide [7,11,13] our understanding of the molecular changes associated with RPE differentiation are still limited
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