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LRP2 gene silencing expression by in vitro shRNA delivery on human iRPE cells

ABSTRACT: To investigate the consequence of LRP2 loss-of-function in RPE cells in non-syndromic myopia, we differentiated IPS cells into iRPE in which the LRP2 gene was knocked out by shRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277911 | GEO | 2025/06/06

REPOSITORIES: GEO

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Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error

Project description:We compare dissected ocular tissues from wild-type and lrp2-/- mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2, a transmembrane receptor, cause ocular enlargement and high-myopia. LRP2 is expressed by the RPE and eye ciliary epithelia, binding many extracellular ligands, including Bmp4 and Shh. Signaling mediated by LRP2 is very context-dependent, and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled, sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia (proper refraction). Using human iPSC-derived RPE, and zebrafish, we demonstrate that BACE sheddase-dependent LRP2 cleavage produces a soluble domain that binds BMP4, inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand-binding receptor forms available: a soluble BMP trap, and a membrane-bound RPE signaling facilitator. By modulating LRP2 cleavage, cells can fine-tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth.

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LRP2 gene silencing expression by in vitro shRNA delivery on human iRPE cells

Project description:LRP2 gene silencing expression by in vitro shRNA delivery on human iRPE cells

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Project description:The retinal pigment epithelium (RPE) is a monolayer of polarized epithelial cells lying between the retina and the choroid/sclera, with a major function in supporting photoreceptor homeostasis, but the role of RPE in eye size regulation is yet to be further explored. it was recently reported that selective deletion of Low density lipoprotein receptor-related protein 2 (Lrp2) in the mouse RPE resulted in large eyes, similar to the phenotypes of Lrp2 whole eye knockout mice, suggesting that the RPE is the tissue in which LRP2 functions. Here, to investigate which downstream pathways of Srebp2 and Lrp2 are responsible for regulating eye growth, we performed RNA-seq analysis with the mouse RPE tissues. Postnatal day 0 (P0) mice were injected with AAV8-Best1-GFP/nSrebp2/ctrl sh/Lrp2 sh1/Lrp2 sh2 viruses to overexpress the constitutively active form of Srebp2 or to knockdown Lrp2 in the RPE specifically. At P14, RPE cells were carefully dissociated for RNA extraction. Via RNA-seq analysis and functional validation, we identified that Bmp2 is the target and the effector of the Srebp2-Lrp2 pathway. Overall, the present study unveils an essential role of RPE in regulating eye size via the Srebp2-Lrp2-Bmp2 signaling pathway, shedding light on the underlying mechanism of eye size control.

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Cell Autonomous Lipid Handling Defects in Stargardt iPS Cell-Derived Retinal Pigment Epithelium Cells

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Project description:Retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSCs), called iRPE, are being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration (AMD). The success of RPE implantation is believed to be linked to the use of biomimetic scaffolds that simulate Bruch’s membrane and aid in its maturation and integration. Current practices widely utilize fibrous scaffolds from synthetic polymers, such as polycaprolactone (PCL), rather than from natural biomaterials. Here, we tested the hypothesis that naturally derived fibrous scaffolds, with their unique translational potential, can provide favorable conditions permissive for the maturation of iRPE in vitro. We show that the culture of iRPE on natural, soy protein-derived nanofibrous scaffolds match the results of similar cells cultured on synthetic PCL nanofibrous scaffolds. The mechanical properties of soy scaffolds better emulate the native Bruch’s membrane, which would likely limit the foreign body reaction triggered by mismatch of host and biomaterial strength. Additionally, soy scaffolds exhibit unique biochemistry that improves biocompatibility and have tunable crosslinking to control degradation rates. Comparative transcriptome analysis demonstrates that iRPE maturation on nanofibrous scaffolds, either natural or synthetic, yielded more consistent outcomes than on other non-fibrous substrates. Taken together, our studies suggest that the maturation of cultured RPE for subsequent clinical applications will benefit from the use of nanofibrous scaffolds derived from natural proteins.

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