Absence of Connexin 43 Results in Smaller Retinas and Arrested, Depolarized Retinal Progenitor Cells in Human Retinal Organoids

Abstract

The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. Lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.

PCDH15 Dual-AAV Gene Therapy for Deafness and Blindness in Usher Syndrome Type 1F

“…The utilization of human retinal organoids derived from human induced pluripotent stem cells has enabled the examination of both retinal development and the study of retinal diseases [53][54][55][56][57][58] . They consist of three nuclear layers and two synaptic layers, replicating the cellular organization observed in the adult retina, and that they exhibit photoreceptor responsiveness to light stimuli and facilitate synaptic transmission of visual information, resulting in light responses in second or third order retinal cells 56 .…”

Targeting Cholesterol Homeostasis Improves Recovery in Experimental Optic Neuritis

“…Retinal organoids were derived from human iPSC [ 30 ]. Human iPSCs were induced to express Brn3a - GFP via CRISPER/Cas9 using a previously described protocol [ 31 ] prior to organoid induction. Starting at day 0, iPSCs were detached from Matrigel-coated plate with mTeSR plus (Stem Cell Technologies, Vancouver, BC, Canada) using TrypLE Express (Invitrogen, Carlsbad, CA, USA) containing 0.05 mg/mL DNase I (Roche, Basel, Switzerland) and 10 μM Y-27632 (Sigma, St. Louis, MO, USA, SML1045-5MG).…”

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders

“…Kanzaki and colleagues [ 29 ] used CRISPR/Cas9 gene editing in healthy iPSCs to knock out KCNJ13 and observed a decrease in the phagocytic activity and expression of phagocytosis-related genes recapitulating the LCA16 phenotype. The same genome editing strategy was also employed to determine the effect of lack of connexin 43 (CX43) on retinal development [ 97 ]. Mutations in the CX43-coding gene, GJA1 , have been associated with oculodentodigital dysplasia (ODDD), including microphthalmia and other ocular defects such as iris atrophy, glaucoma, strabismus and blindness.…”