J Vet Sci.  2017 Mar;18(1):59-65. 10.4142/jvs.2017.18.1.59.

Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells

• Choi SW
• Kim JJ
• Seo MS
• Park SB
• Shin TH
• Shin JH
• Seo Y
• Kim HS
• Kang KS

• KMID: 2412588
• DOI: http://doi.org/10.4142/jvs.2017.18.1.59

Abstract

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. The microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

Keyword

microRNA; miR-410; retinal pigment epithelium; umbilical cord blood-derived mesenchymal stem cells

MeSH Terms

Cell Differentiation/*genetics
Fetal Blood/cytology/metabolism
Gene Expression Regulation, Developmental
Humans
Mesenchymal Stromal Cells/cytology/metabolism
MicroRNAs/genetics/*metabolism
Otx Transcription Factors/*biosynthesis
Phagocytosis
Retinal Pigment Epithelium/metabolism/*physiology
cis-trans-Isomerases/*biosynthesis
MicroRNAs
Otx Transcription Factors
cis-trans-Isomerases

Figure

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  Fig. 1 Putative retinal pigment epithelium (RPE)-specific microRNAs in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). (A) Microarray data indicate the number of microRNAs that are expressed at a higher level in UCB-MSCs than in retina tissue or ARPE-19 cells. Of these, 21 microRNAs belong to both groups. (B) Number of predicted targets for the 21 putative microRNAs determined by using three different target prediction programs; TargetScan, miRanda, and DIANA. (C) Results of real-time RT-PCR analysis of miR-410 expression in human UCB-MSCs (miR ctl), anti-miR-410-treated cells, and the human RPE cell line ARPE-19. ***p < 0.001.

• 

  Fig. 2 Induction of RPE differentiation from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) by miR-410 inhibition. (A) Schematic of the differentiation protocol for UCB-MSCs into retinal pigment epithelium (RPE)-like cells via 2F induction for 9 weeks or by microRNA transfections at days 0, 3, 10 and 17. (B) Conventional RT-PCR measurement of the expression of RPE-specific factors in UCB-MSCs post-induction with 2F and post-treatment with miR control or anti-miR-410 at days 2 and 21. (C) Western blot results for RPE-specific factors Bestrophin and EMMPRIN performed for 2F-induced RPE-like cells and miR-induced RPE-like cells.

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  Fig. 3 Retinal pigment epithelium (RPE)-specific protein expression in RPE-like cells. (A and B) Representative immunocytochemistry images showing that 2F-induced and anti-miR-410-treated RPE-like cells expressed the mature RPE markers Bestrophin and EMMPRIN (both green). Nuclei were counterstained with DAPI. (C) Quantification of Bestrophin- and EMMPRIN-positive cells in miR control, anti-miR-410-treated UCB-MSCs, and ARPE-19 cells. *p < 0.05, ***p < 0.001. Scale bar = 0.2 mm (A and B).

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  Fig. 4 Enhanced phagocytosis of anti-miR-410-treated umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). (A) Representative images of phagocytosis assay results using fluorescent microsphere beads 21 days after transfection of miR control or anti-miR-410 into UCB-MSCs. Treatments of anti-miR-410 in human UCB-MSCs increased the internalization of red fluorescent beads. (B) Quantification of bead phagocytosis level after treatments of anti-miR-410 for 21 days. ***p < 0.001. Scale bars = 50 µm (upper panels), 5 µm (lower panels).

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