David Gamm, MD, PhD – Slide of the Week

David Gamm, MD, PhD - Slide of the Week

Title: Stem cell-derived retinal organoids harboring patient-specific or gene-edited mutations in Interphotoreceptor matrix proteoglycan 2 (IMPG2) fail to extend outer segments or produce an Interphotoreceptor matrix (IPM).

Legend: (A) Retinal organoids (ROs) from a founder human induced pluritpotent stem cell (hiPSC) line containing compound heterozygous mutations in IMPG2 lack visible outer segments (OS) by brightfield microscopy. Immunocytochemistry reveals a thin layer of EC-IMPG2, IC-IMPG2, and IMPG1 expression along the RO surface, along with severely stunted RHO and PRPH2 expression. (B-D) Identical phenotypes are observed in ROs from isogenic IMPG2 knockout (KO/KO) (B) and heterozygous genecorrected (Y254C/GC) (C) hiPSC lines, with the exception that IMPG2 immunostaining is absent in double knockout ROs. (D) Biallelic IMPG2 gene correction (GC/GC) of the founder hiPSC line fully rescues OS and IPM production in ROs as determined by brightfield microscopy and immunocytochemistry. All ROs were differentiated >200 days. RHO = Rhodopsin; PRPH2 = Peripherin 2; CRX = Cone-rod homeobox; scale bars = 20 μm.

Citation:  Mayerl, S. J., Bajgai, S., Ludwig, A. L., Jager, L. D., Williams, B. N., Bacig, C., Stoddard, C., Sinha, D., Philpot, B. D., & Gamm, D. M. (2022). Human retinal organoids harboring IMPG2 mutations exhibit a photoreceptor outer segment phenotype that models advanced retinitis pigmentosa. Stem cell reports, S2213-6711(22)00453-2. Advance online publication. https://doi.org/10.1016/j.stemcr.2022.09.004

Abstract: Interphotoreceptor matrix proteoglycan 2 (IMPG2) mutations cause a severe form of early-onset retinitis pigmentosa (RP) with macular involvement. IMPG2 is expressed by photoreceptors and incorporated into the matrix that surrounds the inner and outer segments (OS) of rods and cones, but the mechanism of IMPG2-RP remains unclear. Loss of Impg2 function in mice produces a mild, late-onset photoreceptor phenotype without the characteristic OS loss that occurs in human patients. We generated retinal organoids (ROs) from patient-derived induced pluripotent stem (iPS) cells and gene-edited embryonic stem cells to model human IMPG2-RP in vitro. All ROs harboring IMPG2 mutations lacked an OS layer, in contrast to isogenic controls. Subsequent protein analyses revealed that this phenotype arises due to a loss of IMPG2 expression or its inability to undergo normal post-translational modifications. We hypothesized that loss of IMPG2 function destabilizes the interphotoreceptor matrix and renders the OS vulnerable to physical stressors, which is accentuated in the tissue culture environment. In support of this mechanism, transplantation of IMPG2 mutant ROs into the protected subretinal space of immunocompromised rodents restored OS production. Beyond providing a robust platform to study IMPG2-RP, this human RO model system may serve a broader role in honing strategies to treat advanced photoreceptor-based diseases.

Keywords: IMPG2; disease modeling; photoreceptors; retinal organoid; retinitis pigmentosa; stem cells.

About the Lab: David Gamm’s laboratory at the Waisman Center uses stem cell technology to investigate the cellular and molecular events that occur during human retinal differentiation and to generate cells for use in human retinal disease modeling and cell-based rescue or replacement strategies. To meet these goals, Gamm utilizes a variety of human cell types, including ES and iPS cells, which have the capacity to mimic retinal development and disease, as well as to delineate the genetic “checkpoints” necessary to produce particular retinal cell types. By understanding the behavior of these cell types in vitro and in vivo, Gamm hopes to optimize strategies to delay or reverse the effects of blinding disorders such as retinitis pigmentosa and age–related macular degeneration.

Investigator: David Gamm, MD, PhD

Contributing Scientist: Steven Mayerl

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