Abstract
Purpose :
To restore vision that has already been lost, RPC need to integrate with the remaining host retina after implantation into subretinal space. We tested whether RPC grown as thick, planar sheets, alone or co-cultured with RPE in their native geometry, could re-populate the outer nuclear layer (ONL) with photoreceptor precursors.
Methods :
H9 human embryonic stem cells were differentiated for 2 weeks to early RPC and seeded on a 60 µm thick, planar scaffold fabricated from gelatin, chondritin sulfate, and hyaluronic acid. Some scaffolds were layered on a monolayer of human fetal RPE (hfRPE) and culture continued for 4 weeks. Metabolic activity was monitored by alamar blue and, retinal markers by quantitative RT-PCR (qPCR) and immunocytochemistry. For transplantation, 30-day-old rd10 mice were used, when the photoreceptor layer would be 60-70 % degenerated. Co-cultured or mono-cultured RPC were engrafted into the subretinal space and analyzed 6 weeks later by mfERG and immunocytochemistry. Antibodies IBA-1 and IL-6 were used to assess evidence of an immune response.
Results :
Co-culture showed higher metabolically active cells (83.3% ± 0.5%) in comparison with mono-culture system. qPCR demonstrated that in presence of hfRPE, stem cells differentiated more efficiently than without hfRPE in vitro with 5 to 15-fold increase in early eye field gene like RAX, SIX3, LHX2, CHX10, 7 to 10-fold increase in photoreceptor markers such as PROX1, Recoverin, CRX, PRPH2 and NR2E3. Six weeks post-implantation, the scaffold was degraded, and TRA-1-85 (human cell marker) positive cells lined the host ONL. There was no evidence of an immune response. TRA-1-85+ cells that lined the outer nuclear layer continued to express recoverin. Pigmented TRA-1-85+ cells were found in the RPE layer. TRA-1-85+ cells with long processes were found in the inner plexiform layer. The mfERG recordings from co-culture mice revealed a modest increase in the P1 wave from 4.86±0.07 nV/deg2 (control) to 7.0±1.0 nV/deg2 (co-culture).
Conclusions :
Planar sheets of RPC can retain differentiated properties after transplantation into the subretinal space. The data provide preliminary evidence that function can be restored in mice with late-stage retinal degeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.