Abstract
Purpose :
Stem cell-derived retinal organoids have uncovered the dynamics of retinal tissue development and pathology caused by inherited retinal degeneration, some of which have suggested potential therapeutics. Yet, the consistency of neuro-retinal architecture is not uniform in suspension cultures as they lack a robust physical scaffold. Therefore, if a physical scaffold that was easy to employ were available, then vitro retinal organoid models may more faithfully recapitulate features of the structure and neuro-retinal maturation found in vivo. Hence, we generated a reproducible and broadly employable synthetic extracellular matrix that improved in vitro retinal organoid generation and maturation.
Methods :
A Rx-GFP mouse embryonic stem cell line was used to create retinal organoids. On day three of differentiation, organoids were embedded in polyethylene glycol (PEG) hydrogels containing reactive groups that could be quickly crosslinked with cell-binding and enzymatically sensitive crosslinking peptides that controlled cella adhesion and gel stiffness, respectively. Samples were fixed, stained, and imaged in seven-day intervals until day 32 of differentiation. Evaluation for the presence of retinal progenitor cells, retinal ganglion cells, and cone photoreceptors were done by IHC staining for Chx10, Brn3a, and Thrb2, respectively.
Results :
Hydrogels containing less than 5% (weight percent) PEG or more than 7% PEG were either too fragile for handling or unable to be synthesized due to solubility limits, respectively. Compared to suspension culture, the spheroid shape was better maintained in the hydrogel model, producing an even laminar structure of neural retina. The 5% PEG hydrogel had more robust Chx10, Brn3a, and Thrb2 staining compared to the 7% PEG hydrogel and sustaining it through the entire differentiation course. Finally, replacement of standard RGD cell-matrix binding peptides with laminin or basement membrane-binding peptides further enhanced neural retina differentiation.
Conclusions :
The creating and screening for a synthetic matrix that embeds retinal organoids improves the efficacy of in vitro retinal stem cell culture. This work can expand to human stem cell-based retinal organoids as well as disease model stem cell lines that not only better understand but also screen for potential therapeutics for retinal diseases with complex genomic heterogeneity.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.