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Eisuke Arai, Bhubanananda Sahu, Lindsay Perusek, Vipul Parmar, Akira Murakami, Akiko Maeda; DHA promotes differentiation of photoreceptor cells in 3D neural retinas. Invest. Ophthalmol. Vis. Sci. 2018;59(9):562.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the effects of docosahexaenoic acid (DHA) on the differentiation of human embryonic stem cells (hESCs) to three-dimensional (3D)-retinal tissues.
We developed a culture method for the differentiation of hESCs to 3D retinal tissues by adding DHA. Expression of genes associated with specific cellular functions of the retina was examined by RT-PCR. Morphology of 3D-retinal tissues was documented on day 120 of the culture and expression of photoreceptor markers was examined by qRT-PCR in the presence or absence of DHA. Furthermore, we performed immunostaining and the number of Rhodopsin positive cells were counted, and thickness of ONL were measured.
hESC aggregates underwent substantial morphological changes and appeared white with a clear cup-like morphology around day 20. RT-PCR detected expression of CRX, Recoverin, RXRγ, CHX10, PROX1, and MITF in hESC-derived 3D-retinal tissues on day 60. Expression of Rhodopsin and BEST1 was appeared by day 150. qRT-PCR demonstrated that mRNA expression of Recoverin and Rhodopsin in 3D-retinal tissues cultured with DHA were elevated compared with those cultured without DHA. Rhodopsin positive cell numbers in retinas differentiated with DHA increased compared with retinas without DHA on day 150, and thickness of ONL in 3D-retinal tissues with DHA was thicker compared without DHA on day 180.
The current study demonstrated that the addition of DHA to culture medium can help promote differentiation of photoreceptor outer segments in vitro and utilization of this methodology may lead to future therapies for patients with blinding diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
(A) Bright field images of 3D-retinal tissues. Scale bars, 50 μm. (B, C) qRT-PCR for Recoverin and Rhodopsin was performed after normalization to the housekeeping gene GAPDH. *P < 0.05; One-way ANOVA with Dunnett`s post hoc comparison. (D-K) Immunohistochemistry was used to identify photoreceptor cells using anti-Recoverin antibody (red; D-G), anti-Rhodopsin 1D4 antibody (red; H-K), and DAPI for nuclei (blue). Scale bars, 30 μm. (L) The number of Rhodopsin positive cells per 500 μm in the middle area of aggregates on day 150. *P < 0.05; Student’s t-test. Error bars indicate mean ± SD (n=3). (M) Thickness of ONL in the middle area of aggregates on day 180. *P < 0.05; Student’s t-test. Error bars indicate mean ± SD (n=3).
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