The gene editing tool CRISPR along with stem cells help scientists create retinal ganglion cells in the lab
Degeneration of retinal ganglion cells often leads to progressive and irreversible vision loss. These cells are the type of nerve cells located within the retina, which transmit visual signals from the eye to the brain. Retinal ganglion cells have limited intrinsic capacity to regenerate and cannot be replaced by new cells. Glaucoma and multiple sclerosis are the most common type of optic neuropathies that lead to vision loss and blindness due to death of retinal ganglion cells.
In a previous blog, we have mentioned that scientists successfully differentiated human embryonic stem cells (HESCs) and human induced pluripotent stem cells (iPSCs) into retinal progenitor and RPE cells. Recently, scientists at Johns Hopkins developed a simple and efficient method to differentiate human stem cells into retinal ganglion cells . Their work not only provides a basic understanding of a facet of optic nerve biology, but also successfully models human retinal ganglion cells in a petri dish that could be used to discover new drugs to treat blindness.
In this study, they combined CRISPR and iPSC technology together to generate retinal ganglion cells. Researchers genetically modified human embryonic stem cells by inserting a fluorescent protein gene into the stem cells' DNA. This stem cell line will express red fluorescence once they differentiate into retinal ganglion cells. This red fluorescent protein would be expressed only if the BRN3B (POU4F2) gene is also expressed, a gene expressed by mature retinal ganglion cells. After the differentiation process, they sorted out retinal ganglion cells from the mixture of cells using a fluorescence-activated cell sorting technique. The sorted pure population showed similar biological and physical properties seen in normal retinal ganglion cells. They also found that adding forskolin, a natural plant chemical, on the first day of differentiation further improved differentiation efficiency. By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope.
Their work lays the groundwork for further experiments on stem cell-derived human retinal ganglion cells, which can be used for drug screening, developmental, and biological studies, as well as cell replacement experiments.
 Valentin M. Sluch, Chung-ha O. Davis, Vinod Ranganathan, Justin M. Kerr, Kellin Krick, Russ Martin, Cynthia A. Berlinicke, Nicholas Marsh-Armstrong, Jeffrey S. Diamond, Hai-Quan Mao, Donald J. Zack.Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. Scientific Reports, 2015; 5: 16595 DOI: 10.1038/srep16595