Abstract
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.
Highlights
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness
BRN3B is expressed in a large majority of RGCs, is RGC specific in the retina, and is relatively restricted in its expression throughout the rest of the body[17,18,19]
We have developed a simple and scalable system for differentiation and purification of human RGCs from Human pluripotent stem cell (hPSC) using a genetically engineered RGC reporter human embryonic stem cells (hESCs) line
Summary
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. We identified the small molecule forskolin as a positive regulator of stem cell differentiation to RGCs. The availability of well-characterized and highly purified human RGCs will provide a useful cell resource for studying human optic nerve biology and disease, a more medically relevant system for drug discovery efforts, and may help in the development of novel cell-based therapies for the optic neuropathies
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