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Laurel T Tainsh, Deepti Singh, SHAO-BIN WANG, Maryam Ghiassi-Nejad, Bo Chen, Ron A Adelman, Lawrence J Rizzolo; Self organization of human induced pluripotent stem cell (iPSC) derived retinal progenitor cells on a 3D scaffold.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1147.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell-derived retinal progenitor cells (RPC) have potential for treating retinal degenerations. Although RPC can form retina-like neurospheres, planar structures would be more appropriate for implantation. We investigated whether RPC could be reconfigured on a planar scaffold.
Human iPSCs were differentiated in DMEM/F12 supplemented with N2 and B27 according to published protocols. After 3 or 8 weeks of differentiation, retinal cups (RCs) were seeded as intact spheres or after dissociation with trypsin onto a scaffold composed of gelatin, hyaluronic acid, and chondroitin sulfate. Differentiation of intact and dissociated RCs on scaffolds was monitored with qPCR at monthly time points for up to 14 weeks as compared to control RCs grown in suspension. Immunohistochemistry, confocal microscopy, and scanning electron microscopy were used to assess cell penetration, proliferation, and differentiation within scaffolds. In some experiments, RPC scaffolds were co-cultured with RPE. Effects on the RPE were assessed by the transepithelial electrical resistance (TER).
Intact RCs attached as clusters and proliferated to invade the scaffold forming distinct subdomains expressing ganglion cell, interneuron, and photoreceptor markers. Intact cell clusters on scaffolds continued to grow in size for up to 6 weeks. Trypsinized RCs populated the scaffold more uniformly reforming as clusters on the surface and laminating within scaffold pores. Scaffolds seeded with dissociated RCs remained uniformly populated for up to 14 weeks. RT-qPCR results showed that both intact and dissociated RCs on scaffolds differentiate along a timeline as well or better than RCs grown in suspension. Intact and dissociated RCs expressed eyefield, ganglion cell, interneuron, and early photoreceptor markers such as recoverin, but did not express the more mature photoreceptor markers opsin and rhodopsin. With co-culture TER increased 3x for the RPE, while PAX6 decreased and OTX2 increased in the RPC.
Both intact and dissociated iPSC derived RCs penetrate, proliferate, and differentiate within HA scaffolds. Dissociating RCs with trypsin before seeding results in a more uniformly populated, laminated, and planar scaffold. Co-culture furthered the maturation of RPE and RPC. Ongoing experiments aim to optimize this approach.
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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