Our eyes have a very limited capacity to regenerate connections lost after injury to the retina. Over a million retinal ganglion cells (RGCs) collectively transmit visual information to various regions of the brain, and death of these RGCs will definitively cause irreversible blindness. Many different approaches to RGC-based therapy are therefore being tried with the hope of restoring vision in people with optic neuropathies.

 

Building a Scalable Approach for Deriving Functional RGCs

At the forefront of this field is Dr. Donald Zack’s team at the Johns Hopkins University School of Medicine and the Stem Cells and Ocular Regenerative Medicine (STORM) Center at the Wilmer Eye Institute. As described in a recent Nature publication, this group has built a CRISPR and cell sorting based, scalable approach for deriving functional RGCs, which recapitulate normal retinal development, from human embryonic stem cells (hESCs).

Retinal organization by hPSC derived retinal ganglion cells.
Figure 1. Retinal organization by hPSC derived retinal ganglion cells. Source: Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line by Sluch, V. M. et al. is licensed under CC BY 4.0.

Using the CRISPR-Cas9 genome editing tool, they inserted a fluorescent protein into the BRN3B gene in human pluripotent stem cells (hPSCs) to mark the RGCs. Then, using the SH800 cell sorter from Sony, they sorted genetically modified and differentiated RGCs into 96-well plates.

The gentle isolation of single cells resulted in hESC-derived RGCs which survived long term, developed neurite networks in culture, and displayed ultrastructural properties of in vivo RGCs. These CRISPR modified cells recapitulated normal retinal development and expressed markers of all retinal lineages.

The availability of such well characterized and highly purified human RGCs will provide a useful cell resource for studying human optic nerve biology and disease, and a more medically relevant system for drug discovery efforts.

 

 


References

Sluch, V. M. et al. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. Sci Rep. 5, 16595; doi: 10.1038/srep16595 (2015). Nature