Cell replacement and visual improvement by CRX-GFP hESC-derived 3D retina organoids in an immunodeficient retinal degenerate (RD) rat

Abstract

Purpose : To study retinal cell differentiation from a Cone-Rod Homeobox (CRX)-GFP expressing human embryonic stem cell line, and visual improvement after transplantation to a rat model of retinal degeneration (RD).

Methods : 3D retina organoids were differentiated from a hESC CRX-GFP cell line (Collin et al. 2016, Stem Cells 34:311–321) using a feeder-free system; protocol similar to Zhong et al. 2014 (Nat. Comm. 5:4047). Organoids were analyzed by qPCR for expression of a variety of neuro-retinal genes. CRX-GFP expression was detected by flow cytometry on dissociated organoids. Retinal sheets were cut from organoids (62-198d of differentiation) and transplanted to the subretinal space of Rho S334ter-3 retinal degeneration (RD) rats. Growth and development of transplants was monitored by high resolution optical coherence tomography (SD-OCT) at regular intervals post-surgery. Visual function was accessed by optokinetic tests (OKT) and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin & eosin (H&E); or processed for immunohistochemistry (IHC) to label human donor cells, retinal cell types and synaptic markers.

Results : The CRX-GFP organoids generated various retinal progenitors in vitro. Flow cytometry of dissociated retinal organoids demonstrates an upregulation of GFP, indicative of CRX expression, with time in culture. After transplantation, organoids developed and matured into various retinal cells and integrated with the degenerating host retina. OCT showed the growth and maturation of the transplants in vivo. Transplanted eyes showed vision improvement by OKT and SC recording. Photoreceptor rosettes in the transplanted rats were positive for the human markers SC121 and Ku80, as well as photoreceptor markers, indicating development of outer segments. In the best cases, transplant photoreceptors aligned in the correct orientation towards host retinal pigment epithelium (RPE) (correct lamination).

Conclusions : Our data shows that a CRX-GFP hESC cell line can generate retinal organoid sheets and after transplanted into the RD rats, can mature into retinal cells, integrate with the host retina and improve visual function. The CRX-GFP hESC cell line provides an excellent opportunity for optimizing future retinal sheet transplantations.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.