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Tina Xia, Deepti Singh, Laurel Tainsh, Ron A Adelman, Lawrence J Rizzolo; Towards culturing retinal progenitor cells as a planar sheet. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4557. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal progenitor cells (RPC) can be successfully differentiated into spherical organoids with retina-like lamination, but planar sheets of retinal tissue would have advantages for experimentation and transplantation. This study explores methods to culture RPC on planar scaffolds with and without laminin-521, a component of the inner limiting membrane.
Retinal cups (RC) were derived from human iPSC and ESC cells using published protocols. After 21 days of differentiation (D21) RC formed containing retinal progenitor cells (RPC). Whole retinal cups, or RC dissociated with trypsin, were seeded on 60µm thick scaffold of gelatin-chondroitin sulfate-hyaluronic acid. For laminin-coating experiments, early stage embryoid bodies were seeded on scaffold coated with 2µg/cm2 of laminin-521. Differentiation was monitored by quantitative RT-PCR and immunofluorescence for expression of early eye field genes and subsequent markers of differentiation.
Whole retinal cups adhered to the scaffold. Some cells invaded the scaffold, but the majority spread as thick clusters along the surface. In comparison, dissociated RC cells also invaded the scaffold, but attachment efficiency was low. On D29 of differentiation, dissociated RC expressed lower levels of early eye field genes PAX6, LHX2, and SIX3. But by D51, gene expression in dissociated and undissociated RC were equivalent. In neither culture did the cells become confluent on the scaffold. For embryoid bodies, there was 57% ±10% (p <0.05) higher attachment on laminin-coated scaffolds compared to non-coated ones. On imaging, cells from embyroid bodies invaded and were uniformly distributed across and within the scaffolds. However, embyroid bodies did not readily differentiate into retinal lineage under these conditions. By D9, stem cells marker, NANOG, was downregulated but OCT4 expression persisted. Expression of early genes RAX, PAX6, and LHX2 were evident at low levels.
Early RC can be dissociated and seeded on scaffolds with low efficiency, and after a delay continued to differentiate. Laminin scaffold coating promotes higher and uniform cell adhesion of embryoid bodies, but retinal differentiation is slower in this condition. Besides being a part of the inner limiting membrane, laminin-521 provides a niche for pluripotent stem cells. In view of these results, current experiments focus on seeding dissociated RC on laminin-coated scaffolds.
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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