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Jie Zhu, Helen Cifuentes, Joseph Reynolds, Deepak A Lamba; Generation of retinal photoreceptors from cGMP-Manufactured Human IPSC line. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Retinal degeneration often results in the loss of photoreceptors, which leads to permanent vision loss. Generating transplantable photoreceptors using human induced pluripotent stem cells (iPSCs) to replace lost or dysfunctional photoreceptors holds a promise to treat a variety of retinal degenerative diseases. Developing effective methods to produce retinal cells including photoreceptors using available cGMP-manufactured human iPSC lines is a critical step for advancing cell replacement therapy to clinical application. This study aims to make transplantable photoreceptors using a cGMP-manufactured iPSC line. The generated retinal cells were tested for their differentiation capability and integration in a host mouse retina.
An iPSC line derived in cGMP-compliant conditions was obtained and used to generate retinal cells via a modified version of our previous directed differentiation protocol (Lamba et al 2006). The cells were characterized via QPCR and immunocytochemistry (ICC) at 6 weeks and 3 months after differentiation. GFP labeled retinal cells were injected into the subretinal space of 4-6 week old recipient mice. The eyes were collected at 2 months post transplantation for analysis by ICC.
iPSCs, generated from CD34+ cord blood cells in a cGMP facility at Lonza Bioscience, were obtained from Dr. Zeng’s lab. They were induced to optic field stage for 1 week using small molecules to inhibit Wnt, BMP and TGFβ signaling pathways along with IGF-1 in culture medium. The cells were then expanded to generate neuro-retina and RPE. Neuro-retinal progenitor cells were manually isolated from RPE and expanded further to a relatively pure population of retinal neurons including differentiated photoreceptors by 3 months of culture based on expression of PAX6, OTX2, TUJ-1, CRX and AIPL1. The cells were then analyzed for their ability to integrate into the ONL layer of host retina in IL2rg null humanized mice. We observed robust integration of photoreceptors with typical mature photoreceptor morphology and presence of Otx2, Recoverin, and Rhodopsin markers in the integrated cells.
This study provides strong evidence that transplantable photoreceptors can be generated from a cGMP-manufactured human iPS cell line which could then be fast-tracked to the clinic.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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