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Lin Cheng, Matthew R. Cring, Markus H Kuehn; Modeling oculodentodigital dysplasia syndrome using human induced pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2878. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Human induced pluripotent stem cells (hiPSC) offer the opportunity to generate lineage-specify cells to investigate mechanism of human diseases which are difficult to study in vivo. Oculodentodigital dysplasia (ODDD) syndrome is caused by mutations in the gene Gap Junction Protein Alpha 1 (GJA1) expressing Connexin43 (CX43). Its symptoms include dental abnormalities, thin nose, syndactyly but also ocular abnormalities such as microphthalmos, iris atrophy, and glaucoma. Here, we established a model of ODDD using hiPSC-derived retinal cups to recapitulate crucial developmental patterns during retinogenesis.
GJA1 was disrupted in hiPSC using CRISPR/Cas9. The derived GJA1-/- lines were characterized by immunofluorescence microscopy, qPCR, and Western blot. GJA1 knockout cell lines and wild-type (WT) hiPSC were differentiated into retinal organoids. Expression levels of neural makers and retinal markers were identified by qPCR and immunolabeling. To prevent potential batch variation among different hiPSC colonies, we used two GJA1 knockout lines for investigation.
Using CRISPR/Cas9 genome editing, we successfully generated multiple hiPSC GJA1 knockout colonies. GJA1−/− hiPSC remain undifferentiated and are morphologically indistinguishable from WT hiPSC. GJA1−/− hiPSC also retain typical hiPSC morphology and have high expression levels of pluripotency markers such as OCT4, SOX2 and NANOG. GJA1−/− hiPSCs did not reveal defects in self-renewal and pluripotency in primed states. However, after 26 days differentiation, GJA1−/− hiPSC fail to generate thick neuroepitheliums in retinal organoids culture, resulting in markedly smaller retinal cups and thinner neural retinas. These retinal cups exhibited reduced expression of the neural marker PAX6. Finally, immunohistochemical analyses revealed a dramatic increase in activated caspase 3 in GJA1−/− retinal cups, indicative of apoptotic cell death.
The loss of GJA1 leads to an ongoing retinal degeneration in the retinal cups. Our approach offers important insights into the retina developmental aspect of ODDD. GJA1−/− hiPSC may be a promising tool for drug screening and gene therapies aimed at treating microphthalmos and could benefit infants or children with ODDD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
GJA1 KO hiPSC remain undifferentiated and have high expression level of pluripotency markers.
GJA1 KO hiPSC generate smaller retinal cups and thinner neural retina.
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