A novel in vitro model of intracellular protein exchange between primary photoreceptors

Abstract

Purpose : Cell replacement therapy is under investigation as a means for rescuing vision. However, findings in photoreceptor (PR) transplantations revealed that donor PRs do not integrate into the host retina but instead exchange cytoplasmic GFP, prompting a need to elucidate the mechanism of this material exchange (ME). To address this, we generated a novel quantitative in vitro model to characterize ME.

Methods : Co-cultures were established using primary retinal dissociates from postnatal 3-5 mouse pups expressing fluorescent genetic reporters (NrlGFP and ROSA^mT/mG) or C57BL/6J stained with Mitotracker far red (MTR) or green (MTG). ME was evaluated as the proportion of double-labelled PRs assessed by flow cytometry and validated by immunohistochemistry using day 0 co-cultures as a baseline. A 6 day transwell co-culture of NrlGFP and ROSA^mT/mG was used to determine whether contact is required for ME. Time-lapse imaging was used to monitor mitochondria movement between cells. Statistical analysis was performed using a two-tailed Student’s t-test.

Results : In co-cultures of NrlGFP and ROSA^mT/mG PRs (n=6), we detected a significant increase in the proportion of double positive cells at day 3 (9.26±1.92%) relative to day 0 (0.61±0.39%, p<0.0005). Incubation of dead cells did not show ME, and no ME was detected when the cells were separated by a transwell (0.72±0.20%, p<00015) (n=3), indicating that this exchange is contact-dependent. In co-cultures of cells stained with MTR and MTG, we observed evidence of ME as early as 16 hours compared to no double positive cells at day 0. Final washes of MTR and MTG were used to control for unincorporated dye and showed minimal staining. Live imaging showed mitochondria transferring via a process from one cell to another.

Conclusions : The results from our study are consistent with our hypothesis that ME requires contact. Evidence of organelle exchange suggests cell fusion or transient cytoplasmic bridge formation as a possible mechanism of ME. Understanding ME will provide valuable insight into the safety and feasibility of cell transplantation as a therapeutic tool.

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