Abstract
Purpose :
Progressive loss of retinal pigment epithelium (RPE) is associated with severe vision impairments in retinal degenerative disorders (retinitis pigmentosa) and age-related macular degeneration (AMD). RPE cells are essential to phagocytose sheds of photoreceptor outer-segment (POS) and absorbs light. Thus, replacing the defective RPE cells with induced pluripotent stem cells (iPSC)-derived RPE cells is a promising and potential strategy for treating retinal degenerative diseases.
Methods :
We generated hiPSC-derived RPE cells for in vivo transplantation study. In the present study, hiPSCs were generated from human epidermal keratinocytes using CytoTneTM-iPS 2.0 Sendai reprogramming kit. The neuroectodermal differentiation of RPE was performed by monolayer culture embryoid bodies in vitronectin coated plates containing retinal differentiation medium supplemented with B27. The differentiated RPE cells were characterized using immunostaining, Western blot, and PCR. Furthermore, suspension of GFP labeled hiPSC-RPE cells were injected in the subretinal spaces of 12 RCS rats.
Results :
hiPSCs were positive for pluripotency markers like Oct4, Sox2, Nanog, Tra1-60, and SSEA4. Furthermore, the differentiated RPE showed characteristic flat hexagonal morphology that expressed markers for tight junctions (ZO1), and RPE cells (MITF, CRALBP, Bestrophin, and RPE65). After eight weeks of differentiation, the pigmentation was observed in RPE cells analyzed using a transmission electron microscope (TEM). In a long-term culture, the formation of RPE fluid-filled sac was observed and a layer of sac expressed marker, such as Bestrophin. The phagocytic activity of RPE was evident as phagocytose of latex beads. Furthermore, in vivo study of transplantation showed the improvement of visual function apparent in electroretinogram (ERG) analysis. This indicates the rescue of photoreceptor loss in retinal degenerative rats, but validation with histological assessments are required to confirm the improvement.
Conclusions :
To conclude, feeder-free hiPSC was generated from human keratinocytes differentiated into functional RPE cells. Also, the implantation of hiPSC-RPE cells showed transient improvement of visual acuity in RCS rats.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.