Researchers Grow Retinal Ganglion Cells in the Laboratory


Researchers from laboratory of Donald Zack at The Johns Hopkins University in Baltimore, Maryland have used genome editing methods to efficiently differentiate human pluripotent stem cells into retinal ganglion cells. Retinal ganglion cells are found in the retina that and helps transmit visual signals from the eye to the brain. Abnormalities or death of ganglion cells can cause vision loss, and conditions such as glaucoma and multiple sclerosis can wreak havoc on ganglion cells.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” said Zack, who is the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

Published in the journal Scientific Reports, Zack and his team genetically modified a line of human embryonic stem cells so that they would fluoresce once they differentiated into retinal ganglion cells. Then they used these cells to develop new differentiation methods and characterize the resulting cells.

To genetically modify their cells, Zack and others used the CRISPR-Cas9 system. CRISPR stands for “clustered regularly interspaced short palindromic repeats” and these are short segments DNA, which are found in bacteria, contain short repeated sequences. Following each repeated sequence is a short spacer that usually comes from previous exposures to a bacterial virus or plasmid. Bacteria use the CRISPR/Cas system as a kind of immune system that prevents cells from being invaded by foreign DNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes.

When bacteria are invaded by a virus, the particular Cas nucleases capture the viral DNA, cut it and insert it into the CRISPR array. When the bacterial cell is infected by a virus, an RNA is transcribed from the CRISPR array called the crRNA. This crRNA then hybridizes with the invading DNA or RNA and the double-stranded RNA or DNA/RNA hybrid is degraded by Cas proteins.

The CRISPR/Cas system is a useful laboratory tool for gene editing or adding, disrupting or changing the sequences of particular genes. If Cas9 and the appropriate crRNA are delivered into cells, you can cut a genome almost anywhere. CRISPR has a huge number of potential applications.

Zack and his group used the CRISPR/Cas system to insert a fluorescent protein gene into the DNA of their stem cells line. This red fluorescent protein would be expressed if a gene called BRN3B (POU4F2) was also expressed. BRN3B is expressed by mature retinal ganglion cells. Therefore, once these cells differentiated into retinal ganglion cells, they would glow red when viewed with a fluorescence microscope.

After differentiating their cells, Zack and his coworkers used a technique called fluorescence-activated cell sorting to isolate fully differentiated cells from other cells. The pure cell culture contained cells that displayed the biological and physical properties observed in retinal ganglion cells produced naturally, according to Zack.

As an added bonus, Valentin Sluch, a former graduate student in Zack’s laboratory, and her colleagues discovered that soaking the pluripotent stem cells in a chemical called “forskolin” at the commencement of the differentiation protocol significantly improved the efficiency of differentiation. Forskolin is a labdane diterpene found in the roots of the Indian Coleus plant (Coleus forskohlii), which belongs to the mint family.  It is used by some people as a weight loss supplement by some people.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” said Sluch, who is now a postdoctoral scholar working at Novartis. Sluch continued, “I was very excited when it first worked. I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for Multiple Sclerosis, Zack is collaborating with Dr. Peter Calabresi, professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).