MicroRNA profiling reveals important functions of miR-125b and let-7a during human retinal pigment epithelial cell differentiation

https://doi.org/10.1016/j.exer.2019.107883Get rights and content

Highlights

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    MicroRNAs are dynamically regulated during hESC differentiation into RPE.

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    miR-204, -125b, −211, and let-7 family are the most abundant miRNAs in RPE.

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    miR-204, -125b, −211, and let-7 miRNAs constitute more than half of the RPE miRNome.

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    miR-125b-5p and let-7a-5p support the maintenance of RPE by upregulating MITF.

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    miR-125b-5p and let-7a-5p enhance the differentiation of RPE from hESCs.

Abstract

Retinal pigment epithelial (RPE) cells are indispensable for eye organogenesis and vision. To realize the therapeutic potential of in vitro-generated RPE cells for cell-replacement therapy of RPE-related retinopathies, molecular mechanisms of RPE specification and maturation need to be investigated. So far, many attempts have been made to decipher the regulatory networks involved in the differentiation of human pluripotent stem cells into RPE cells. Here, we exploited a highly-efficient RPE differentiation protocol to determine global expression patterns of microRNAs (miRNAs) during human embryonic stem cell (hESC) differentiation into RPE using small RNA sequencing. Our results revealed a significant downregulation of pluripotency-associated miRNAs along with a significant upregulation of RPE-associated miRNAs in differentiating cells. Our functional analyses indicated that two RPE-enriched miRNAs (i.e. miR-125b and let-7a) could promote RPE fate at the expense of neural fate during RPE differentiation. Taken together, these mechanistic interrogations might shed light on a better understanding of RPE cell development and provide insights for the future application of these cells in regenerative medicine.

Introduction

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells interposed between the choriocapillaris and the neural retina. RPE cells direct interact with photoreceptors and contribute to various cellular and metabolic processes of photoreceptors, including visual cycle, phagocytosis of rod outer segments, and secretion of essential neurotrophic and growth factors. Close developmental and physiological relationships between these cells and neighboring neural retinal cells, make RPE cells an indispensable part of the ocular tissue, essential for retinal development and physiological hemostasis (Strauss, 2005).

From a developmental point of view, both RPE and photoreceptors originate from bi-potent neuroepithelial cells constituting the optic vesicle. During eye development, the optic vesicle is formed as a result of eye field bilateral evagination at the end stages of neural tube formation. While initially the entire optic vesicle is comprised of bi-potential cells expressing the major RPE transcription factor, microphthalmia-associated transcription factor (MITF), subsequent endocrine and autocrine signals emanating from neuroepithelium or surrounding tissues, restrict their potency and specify their fate (Galy et al., 2002).

Damage or dysfunction of RPE is associated with various sight-threatening retinopathies such as age-related macular degeneration (AMD), Best's disease, Stargardt's disease, or subtypes of retinitis pigmentosa (Colijn et al., 2017; Strauss, 2005). So far, conventional treatment of aforementioned retinopathies is of limited therapeutic value. Therefore, extensive investigations have been done to develop more effective treatment regimens. Stem-cell-based therapeutic approaches aim to restore vision through transplantation of RPE cells, derived from human pluripotent stem cells (hPSCs) [i.e., human embryonic stem cell (hESC) and induced pluripotent stem cells (hiPSCs)]. Several clinical trials are currently underway to restore RPE numbers ((Mandai et al., 2017; Schwartz et al., 2015; Song and Bharti, 2016; Song et al., 2015).

Despite encouraging progress in efficient generation of functional RPE cells in vitro, the field encounters several problems such as limited visual improvement, impaired RPE cell survival and function, and insufficient cell integration (Gullapalli et al., 2005; Thumann et al., 2009). This might be due, in part, to the lack of a comprehensive understanding of gene regulatory networks determining RPE cells' identity, physiological functions, and differentiation (Amram et al., 2017). Hence, several studies have attempted to decipher the underlying molecular regulatory networks of RPE cells during stem cell differentiation focusing on mRNA-, epigenetic-, and microRNA (miRNA)-based mechanisms (Liao et al., 2010; Liu et al., 2014; Wang et al., 2010).

miRNAs are ~22-nucleotide regulatory RNAs that control gene expression at the post-transcriptional level (Carthew and Sontheimer, 2009). miRNAs are assumed to target at least up to one-third of all mammalian transcripts, thereby modulating virtually all cellular pathways and developmental processes including cell survival, proliferation, stem cell self-renewal (Hassani et al., 2019; Hwang and Mendell, 2007; Moradi et al., 2018), differentiation, and cell state transitions (Moradi et al., 2014). Given the pleiotropic nature of miRNA regulatory functions in which one miRNA can target tens to hundreds of transcripts, deregulation of miRNAs or miRNA-processing enzymes during development and diseases can lead to diverse phenotypes (Olson, 2014; Pinter and Hindges, 2010).

Studies on Dicer1 and Dgcr8 knockout mice have demonstrated the essential roles of miRNAs in the regulation of ocular development. As such, severe developmental defects of the neural retina, lens, and cornea were observed in cells lacking Dicer1 (Damiani et al., 2008; Georgi and Reh, 2010; Pinter and Hindges, 2010). In addition, various degrees of microphthalmia were observed depending on the timing of Dicer knockout. Interestingly, although RPE cells defective for Dicer1 and Dgcr8, display apparently normal differentiation and gross typical morphology, they suffer from severe defects resulting in photoreceptor cell death over time. In fact, pronounced failure of the visual cycle, impaired phagocytosomes and clathrin-coated vesicles, and defects in cell adhesion were observed in Dicer1 knockout mice (Ohana et al., 2015; Sundermeier et al., 2017). Moreover, Dicer1 downregulation has been documented in patients with dry AMD (Kaneko et al., 2011) indicating essential functions of miRNAs in the physiology and pathology of RPE cells (Ohana et al., 2015; Sundermeier et al., 2017). So far, a thorough understanding of the expression and functional significance of individual miRNAs during the development of RPE cells from pluripotent stem cells is critically missing.

In this study, we determined the expression profile of miRNA during RPE differentiation from hESCs, using small RNA next-generation sequencing. Unlike previous RPE miRNA profiling over the course of hESC differentiation, in which, samples were obtained from spontaneously differentiating cells with a high degree of heterogeneity in the mid stages (Hu et al., 2012), we utilized a differentiation protocol that directs the pluripotent stem cells toward the desired RPE fate in a controlled and efficient manner (Zahabi et al., 2012). We observed that miRNAs were dynamically expressed during the hESC-RPE transition. As expected, miRNAs associated with pluripotency were downregulated while others, associated with differentiated RPE fate were upregulated. We found that the let-7 family of miRNAs together with miR-125b-5p were among the most abundantly expressed miRNAs in differentiating and terminally-differentiated RPE cells. Our functional analyses revealed that let-7 and miR-125b-5p enhanced maturation of RPE cells and generated a more robust RPE state.

Section snippets

hESC culture

hESCs (Royan H6) were cultured on Matrigel (1:30, Sigma-Aldrich) under feeder-free conditions as previously described (Totonchi et al., 2010). Briefly, cells were cultivated in Dulbecco's Modified Eagle's Medium supplemented with 20% knockout serum replacement (KOSR), 2 mM L-glutamine, 1% nonessential amino acids (NEAAs), 1% penicillin and streptomycin, 1% insulin-transferrin-selenium (all from Gibco), 0.1 mM β-mercaptoethanol (β-ME, Sigma-Aldrich), and 100 ng/mL basic fibroblast growth factor

Differentiation of RPE cells from hESCs

In order to investigate the underlying mechanisms of RPE generation from ESCs at the miRNA level, we utilized our previously described efficient protocol for direct and step-wise RPE differentiation from PSCs (Zahabi et al., 2012). The implemented protocol (Fig. 1A) entails BMP inhibition by recombinant noggin treatment upon which ESCs (Fig. 1B) are efficiently driven towards anterior neural ectoderm fate. Approximately 18 days post-differentiation, large populations of neural tube-like

Discussion

RPE cells are the derivatives of the optic neuroepithelium which initially occupies a single domain, the eye field, spanning the midline of anterior neuroectoderm. With the advent of high-throughput sequencing, system-wide evaluations of cellular mechanisms involved in RPE cell differentiation and physiology have become feasible. Several studies have been conducted to decipher the miRNome and transcriptome of the retinal cells under different developmental and diseased conditions (Karali et

Author disclosure statement

The authors declare that they have no conflicts of interest.

Acknowledgments

We are grateful to Dr. Chitsaz lab, Colorado state University, for providing us with data storage space for NGS data. We also thank Dr. Marzieh Ebrahimi for providing miR-204–5p and miR-211–5p primers. We would also like to thank other colleagues at Royan Institute for Stem Cell Biology and Technology for constructive discussions. This work was supported by a grant from Royan Institute, and National lnstitute for Medical Research and Development, NIMAD, Grant No. 976881) to H.B.

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