HARVARD DEPARTMENT OF OPHTHALMOLOGY
Category : Basic and Translational Research Candidate : Jonathan Soucy Poster #: B20
Chemokine-directed Migration Improves the Structural Integration of Human Stem Cell-derived Retinal Neurons
Jonathan Soucy , Petr Baranov
Purpose : Cell transplantation has been proposed to restore retinal ganglion cell ( RGC ) populations lost in glaucoma and other optic neuropathies . While donor RGCs can survive in host retinas following transplantation , cell survival does not equate to vision restoration , and poor integration remains a significant challenge for successful RGC replacement . The inner limiting membrane ( ILM ) has recently been identified as a major barrier to donor cell migration into the ganglion cell layer ( GCL ) following intravitreal delivery . Therefore , to overcome this limitation , we delivered donor RGC subretinally , then directed their migration towards the GCL by engineering chemokine gradients across the retina .
Methods : To identify our lead candidate for RGC recruitment , we studied the migration of human RGC in response to different chemokine gradients in vitro . For this and other experiments , RGCs were differentiated from pluripotent stem cells by using retinal organoid cultures . After identifying SDF1 as the most potent chemokine for RGC recruitment , we transplanted human RGCs subretinally and delivered recombinant SDF1 protein intravitreally to establish a chemokine gradient across the retina . To investigate the mechanisms of chemoattraction and the modes of donor RGC migration , we applied small-molecule inhibitors to donor RGCs prior to transplantation for 1 ) the CXCR4 receptor – AMD3100 , 2 ) somal translocation ( ST ) – CK666 , and 3 ) multipolar migration ( MP ) – roscovitine . Three days post-transplantation , retinas were stained for donor and host neurons and evaluated by tracking the position of each donor RGC in 3D reconstructions of retinal flat mounts .
Results : Treatment with SDF1 increased donor RGC integration into the GCL from 16 ± 11 % to 44 ± 11 %. We confirmed the direct effect of SDF1 on donor cells by inhibiting in response to SDF1 by blocking SDF1-CXCR4 binding in donor cells with AMD3100 ( 17 ± 5.9 %). Further , inhibiting MP migration significantly limits the percentage of donor RGCs that migrate to the GCL in response to SDF1 ( No inhibition : 45 ± 15 %; MP inhibition : 20 ± 3.7 %), whereas inhibiting ST does not affect donor RGC migration ( ST inhibition : 47 ± 12 %) – indicating donor RGCs migrate via MP in vivo . The inhibition of migration did not affect survival , and each migration pattern was confirmed with an in vitro time-lapse study .
Conclusions : Altogether , our studies confirm that donor cell behavior is controllable by modulating the tissue microenvironment and that SDF1 gradients across the host retina improve the structural integration of donor RGCs . Furthermore , our results show that MP migration is the primary mode of migration for surviving donor human RGCs in the retina and that SDF1 acts through the CXCR4 receptor – suggesting the possibility that RGCs selected for these features before transplantation will improve donor cell integration .