Abstract
Purpose :
Neurofibromatosis type 1 (NF) is a pediatric neoplasia of the nervous system, caused by germline mutations of the neurofibromin gene. Among characteristic pathological phenotypes, the development of optic pathway gliomas (OPGs) affects visual acuity or cause blindness among other ophthalmic features in symptomatic patients. Although transgenic rodent models of NF1 OPGs have helped elucidate disease pathogenesis, significant differences exist in the retina and optic nerves of rodents and primates, including differences in NF1 disease pathology. To address this shortcoming, we have developed a human induced pluripotent stem cell (iPSC) model that mimics many features of the optic nerve and supporting glial cells as a novel tool to characterize OPG-associated clinical heterogeneity and prognostic markers at cellular level.
Methods :
NF1-relevant single nucleotide polymorphisms (SNPs) were introduced into lines of iPSCs using CRISPR editing, resulting in paired disease models and isogenic controls. These cell lines were then differentiated to yield retinal organoids following established procedures, and retinal ganglion cells (RGCs) were purified and characterized for changes associated with the NF1 gene variants. Assessment of RGC changes include morphological alterations assessed by neurite complexity as well as changes in gene and protein expression.
Results :
We have successfully generated lines of iPSCs with NF1-associated gene variants via CRISPR/Cas9 gene editing, and further analyses of these cell lines assess for the possibility of off-target effects of gene editing. These cell lines were also edited to express an RGC-specific mNeonGreen reporter for RGC identification, as well as the mThy1.2 cell surface antigen for subsequent RGC purification. Retinal organoid differentiation and subsequent RGC purification results in highly enriched population of RGCs from all cell lines tests. Experiments in progress will further assess changes in RGC morphological features as well as functional properties.
Conclusions :
Taken together, these results demonstrate the generation of human iPS cell lines with patient-relevant NF1 gene variants as a disease model for neurofibromatosis type 1, as well as the use of these cells to begin to explore features associated with the OPG phenotype.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.