3D retina organoids produced from CRX-GFP human embryonic stem cells (hESCs) processed into retinal sheets and transplanted into immunodeficient retinal degenerate (RD) rats

Abstract

Purpose : To demonstrate retinal and photoreceptor differentiation from a Cone-Rod Homeobox (CRX)-GFP cell line in vitro (feeder free) and in vivo 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 after Zhong et al. 2014 (Nat. Comm. 5:4047). Retinal sheets were cut from organoids and prepared for transplantation. Remainder “pieces” of processed organoids were analyzed by qPCR for a variety of neuro-retinal genes. Flow cytometry on dissociated organoids at various time points allowed for the detection of a CRX-GFP+ population. After being loaded into a patented implantation tool, the tissue was deposited subretinally into 55d-old immunodeficient RCS rats (n=5). Growth and development of transplants was monitored by ocular coherence tomography (OCT) at regular intervals post-surgery. Cryostat sections of transplanted eyes were analyzed by immunohistochemistry for donor and retinal markers.

Results : Quantitative PCR using a custom retinal array validated that the CRX-GFP organoids contained the various progenitors and mature cell types critical to in vivo photoreceptor development and integration with the host. In culture, the retina organoids spontaneously generate various retinal progenitors and sub-populations as determined by qPCR and histology. Flow cytometry of dissociated retinal organoids demonstrates an upregulation of GFP, indicative of CRX expression, with time in culture. OCT scans monitored the growth and maturation of the transplanted sheets. Photoreceptor rosettes within the transplant were positive for the human markers SC121 and Ku80, as well as photoreceptor markers recoverin and rhodopsin.

Conclusions : Our data shows that a CRX-GFP hESC cell line can generate retinal tissue through feeder-free 3D organoid culture. The resulting retinal organoids can be further processed into transplantable sheets. The CRX-GFP hESC cell line is capable of generating new photoreceptors within the RCS RD retina and provides an excellent opportunity for optimizing future retinal sheet transplantations.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.