Abstract
Purpose :
Stem cell transplantation may serve as a novel therapeutic strategy to address retinal degenerative diseases by providing neuroprotection and regeneration. However, cellular engraftment into the retinal ganglion cell (RGC) layer following intravitreal delivery is almost negligible. The retina is a well-guarded structure and the inner limiting membrane (ILM) acts as the physical barrier between the vitreous body and the neural retina. The ILM is formed by the end feet of Muller glia, astrocytes and basement membrane. This study aimed to modulate the ILM in a transgenic mouse model by targeting adherens junction molecule N-cadherin (N-cad) expressed in Muller glia in order to evaluate transplanted stem cell engraftment and viability.
Methods :
A human induced pluripotent stem cell (hiPSC) line labeled with green fluorescence protein (GFP) underwent retinal differentiation by an established retinal organoid strategy and RGC maturation protocols. Retinal organoids were dissociated and GFP-labeled RGC-committed progenitors were isolated by magnetic activated cell sorting (MACS) for Thy1 positive cells. A transgenic mouse model expressing Cre recombinase (CreERT) under the control of solute carrier family 1 (Slc1a3)-glial high affinity glutamate transporter (GLAST) promoter was crossed with a floxed target strain of N-cad. Following tamoxifen induction in the resulting GLAST-CreER/N-cadflox transgenic mice, hiPSC-derived RGC-progenitors were injected intravitreally into the eyes. After 8 days, the retinas were collected for RT-PCR and western blot (WB) analysis or dissected for flat-mount and sectioning. Immunofluorescence (IF) staining was performed to evaluate engraftment of the hiPSC-derived RGC-progenitors.
Results :
RT-PCR and WB analysis confirmed that upon tamoxifen induction, N-cad was knocked-down. IF analysis showed disruption of N-cad in Muller glia. Furthermore, at 8 days post-delivery, GFP-labeled hiPSC-derived RGC-progenitors were specifically detected penetrating into the modulated neural retina. Transplanted cells in animals with ILM modulation had higher viability and morphological evidence of engraftment with host tissues.
Conclusions :
The temporary disruption of adherens junctions present in Muller glia forming the ILM could provide valuable insights into engraftment mechanisms of stem cells and augment their regenerative capabilities.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.