hESC-derived retina organoid sheet transplants develop photoreceptors, connect with the host retina and improve visual function in immunodeficient RCS rats

Abstract

Purpose : To study whether sheets from human embryonic stem cells (hESC)-derived retinal organoids can survive and improve visual function after transplantation to the subretinal space of a slow retinal degeneration rat model, immunodeficient RCS rats.

Methods : CSC14 (NIH line 0284) hESCs were differentiated into retina organoids (modification of Zhong et al. 2014. Nature Communications; McLelland et al. 2018, IOVS) and characterized by immunohistochemistry (IHC) and qPCR. Sheets dissected from retina organoids (differentiation day 30-70) were transplanted to the subretinal space of nude RCS rats (P44-56). Optical Coherence Tomography (OCT) monitored transplant development in vivo. Visual function was assessed by optokinetic testing (OKT), electroretinogram (ERG) and electrophysiological recording in the superior colliculus (SC recording) at 6-10 mo. post surgery (MPS), and compared with age-matched RCS controls. Tissue sections were analyzed by histology and confocal immunohistochemistry (IHC) for human donor, retinal and synaptic markers.

Results : IHC of retina organoids showed early lamination and developing retinal progenitors. qPCR analysis indicated an expression profile closest to that of human fetal retina; primarily comprised of various retinal progenitor markers (CHX10+, CRX+, NRL+, LHX2+). OCT imaging revealed the transplant growth in the subretinal space and the development of rosettes. At 2 MPS, ERG recording of transplanted eyes revealed increased B-waves to scotopic and photopic stimulation compared to RCS controls, but not later (4-6 MPS). However, OKT and SC recording showed significant vision improvement that was maintained at 6-10 MPS. Transplants developed a rosetted morphology, with a distinct outer nuclear layer and photoreceptor outer segments located in the center of rosettes. Human-specific label indicated donor cell migration, and extension of processes into the host retina, with increased synaptophysin immunoreactivity in the host IPL adjacent to the transplants. Photoreceptors in the center of rosettes stained for rod and cone markers.

Conclusions : hESCs derived retina organoids transplanted to the subretinal space of immunodeficient RCS s matured, developed photoreceptors with outer segments, integrated into the host retina, and improved visual function by both rescue and replacement effects.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

OKT testing results