Effect of immunosuppression on hESC-derived retina organoids in vitro and in vivo

Abstract

Purpose : The effect of immunosuppression on the development of human embryonic stem cell (hESC) derived retina organoids (ROs) produced by a scalable cGMP compatible process was studied in vitro and after transplantation to the subretinal space of immunocompetent retinal degeneration (RD) rats.

Methods : A Working Cell Bank (WCB) of CSC-14 hESCs (NIH 0284) was established using a scalable cGMP compatible process. ROs differentiated from hESCs were characterized by immunohistochemistry (IHC), flow cytometry, qPCR and a two-way mixed lymphocyte reaction (MLR). Functional and structural imaging of organoids was obtained using fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSpec). Retina organoid sheets were transplanted to the RD hosts and monitored by Optical Coherence Tomography (OCT). Immunosuppressants (20-30mg Tacrolimus [TAC] pellet implant in combination with oral mycophenolate mofetil [MMF]) were applied to the rats. TAC levels in blood were determined by LC-MS. Cytostatic effects on target lymphocyte populations were evaluated by flow cytometry. Visual function was accessed by optokinetic tests and superior colliculus electrophysiology. Sections through transplants were stained with hematoxylin & eosin (H&E) and IHC. The effect of TAC (3 ng/ml)) and MPA (0.5 µg/ml) on retinal organoids was tested by FLIM after 1 and 4 weeks exposure.

Results : The WCB of hESCs meets the FDA requirements. In vitro immunogenicity tests showed that ROs are not likely to induce an immune response. IHC of ROs shows early lamination and development of retinal cell progenitors. At 2-3 mo. of differentiation, organoids switched their metabolic status from more glycolytic to more oxidative which remained stable at 4 months. Immunosuppressants TAC and MPA showed no influence on the metabolic activity of retinal organoids after 1 and 4 weeks of exposure. OCT revealed transplant development and photoreceptor rosettes. The transplants developed different retinal cells including photoreceptors; and integrated with the host retina. Therapeutic levels of immunosuppressant remained in the blood and helped transplants survive in the host.

Conclusions : Retina organoids matured and developed photoreceptors in long-term culture and after transplantation. Immunosuppressants did not measurably alter the metabolic status of organoids, and prevented rejection of retinal organoid transplants.

This is a 2021 ARVO Annual Meeting abstract.