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Chad Alan Galloway, Sonal Dalvi, Lauren Winschel, Leslie MacDonald, Ruchira Singh; hiPSC-derived retinal cell model of juvenile neuronal ceroid lipofuscinosis (JNCL, CLN3). Invest. Ophthalmol. Vis. Sci. 2017;58(8):294.
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© ARVO (1962-2015); The Authors (2016-present)
Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease, CLN3) leads to progressive neurological dysfunction and retinal degeneration. Histopathologic studies showed accumulation of autofluorescent lipopigment in retinal neurons and retinal pigment epithelium (RPE) and degeneration of multiple retinal cell layers in JNCL. However, the precise involvement of a specific retinal cell type in the disease and the underlying disease mechanism responsible for vision loss is not known. The purpose of this study is to determine the morphological and molecular consequences of JNCL in a patient-derived human induced pluripotent stem cell (hiPSC) model of JNCL.
hiPSCs were generated by reprogramming of fibroblasts from JNCL patients (1.02 kb deletion in CLN3) and unaffected family members. Pluripotency confirmed JNCL patient and control hiPSCs were differentiated to RPE and optic vesicles (OVs). OVs were harvested at day 20, 35, 55, and 90 coordinate with critical in vivo developmental stages of the eye. Immunocytochemistry, qRT-PCR and Western blotting were utilized to determine 1) the spatiotemporal expression of CLN3, 2) autofluorescent lipopigment accumulation and 3) the differentiation and viability of neural retina (NR) cell types and RPE in patient vs. control cultures.
CLN3 gene and protein expression peaked at the time point of OV formation (D20) and remained similar between control and patient hiPSC OVs at all time points evaluated. An increased accumulation of autofluorescent material, corresponding to lipopigment excitation/emission spectrum, was seen in patient OVs and RPE. While, neural retinal progenitor cells gave rise to all NR cell types in control and patient OVs, gene and protein expression analysis suggested the specific loss of ganglion, bipolar and photoreceptor cells at 90 days in patient-derived OVs. In contrast expression of glial markers (S100, GFAP) was increased in patient-OVs compared to control OVs.
hiPSC-derived retinal model of JNCL displays several important features consistent with the disease pathology including 1) autofluorescent material accumulation in NR and RPE, 2) decreased expression of neural retina markers in photoreceptors, bipolar cells and ganglion cells and 3) increased expression of glial markers. Furthermore, this hiPSC model of JNCL suggests that CLN3 loss of function but not loss of expression leads to the disease pathology.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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