Co-treatment with FasL and Annexin modulates host microglia after donor RGC transplantation and improves the outcome


Volha Malechka, Jonathan Soucy, Emil Kriukov, John Dayron Rivera, Petr Y Baranov | 2023


Purpose : The stem cell-derived retinal ganglion cell (RGC) transplantation shows the strong evidence of RGC integration into the retina in animal models. The major challenge of RGC is the survival of donor RGC cells in the host retina. We have shown previously that host retinal microglia is the major contributor to the acceptance and survival of donor RGCs. The aim of this study was to enhance the acceptance and improve the survival of donor RGCs in the host environment by modulating host retinal microglia using pharmacological approaches.

Methods : Brn3b-/- mice were used for RGC transplantation studies. The donor RGCs were formulated, pretreated with Fas Ligand (FasL) and annexin, and 1x104 of viable cells were injected into one eye of each mouse by subretinal delivery. Retinas were collected 3 days after transplantation and stained for donor RGCs (mCherry) and microglia/macrophages (Iba1). Structural integration of donor RGCs was evaluated in retinal flat mounts by confocal microscopy. To assess RGC-microglia interaction, we used single-cell RNA-seq data from human fetal (FD59, FD82, and FD125) and adult retina.

Results : Pretreatment of donor RGCs with FasL alone, both FasL and annexin before subretinal delivery resulted in improvement of donor RGC survival rate by 60% compared to control. Pretreatment of donor RGCs with FasL and annexin resulted in a decreased activation of microglia cells in the host environment compared to the group of donor RGCs pretreated with FasL alone by 40%. We quantified the expression level of LRP1, GAS6, TULP1, MFGE8, ANXA1, CALR, C1QA, C3, GLA, PROS1, PTDSS1 genes as “eat-me” signal and CD24, CD47 genes as “do-not-eat-me” signal. The most dramatic changes were observed for CD24 resulting in a sequential decrease from 19.67 to 0.018, and in increase from 0.12 to 0.99 for CD47 expression levels.

Conclusions : Our findings confirm that FasL and annexin may activate pro-survival pathways and create an environment for survival of donor RGCs after transplantation.