Abstract
Purpose :
We previously reported the use of human ES/iPS cell-derived retinas for xenotransplantation (Iraha et al., Stem Cell Reports, 2018; Tu et al., EBioMedicine, 2018). While graft photoreceptors mature and form the outer nuclear layer-like structure in the host retina, the lack of robust light response detected in the host retinal ganglion cells (RGCs) suggests an inefficiency of graft-host contact, likely due to the presence of graft inner neurons in part. To address this possibility, we established a genetically engineered hES cell line that differentiates into hESC-retinas with less bipolar cells after maturation in the host (Yamasaki et al., IOVS, 2018). Here we further examined the graft-originated light responses of the host retinas, showing the potential graft-host synaptic connectivity.
Methods :
The hESC-retinas were stably differentiated from human CRX-GFP ESC lines, with or without ISLET1 knockout (KO), and transplanted to immunodeficient rats with retinal degeneration at 4-5 months of age. Graft maturation and integration were confirmed by immunohistochemistry 6 months later, and graft function was examined by multi-electrode array (MEA) recording at age of 1 year or more. Field potentials as micro-electroretinogram (mERG) and RGC spikes to full-field light stimulation were detected and analyzed in accordance with the graft positions. Opsinamide was used to suppress the melanopsin-driven RGC intrinsic photosensitivity, and L-AP4 was applied to confirm the mGluR6-mediated photoreceptor-bipolar cell synaptic transmission.
Results :
Both mERG b-waves (ON bipolar cell depolarization) and host RGC responses were diminished in the presence of L-AP4. Along with a reduced bipolar cell number in the KO hESC-grafts, mERG b-waves were less detected in the transplanted retinas. In contrast, RGC responses were steadily detected in the rat retinas with KO-transplants, consistent with the observation of increased graft photoreceptor-host bipolar cell contact. These results together indicate the light responses originated from graft photoreceptors and were transmitted via graft and/or host bipolar cells.
Conclusions :
Genetically engineered hESC-retinas with less bipolar cells prove to be feasible for retinal reconstruction by supplying mature, light-sensitive photoreceptors and driving potential synaptic transmission to the host inner retinal neurons.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.