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xitiz chamling, Valentin Sluch, Karl J Wahlin, Donald J Zack; Screen for Small Molecules that Promote Myelination of Human Stem Cell Derived Oligodenrocytes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5903.
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© ARVO (1962-2015); The Authors (2016-present)
Multiple Sclerosis, a neurodegenerative disease whose pathogenesis involves immune-mediated demyelination, is a leading cause of optic neuritis. Currently, there are no clinically approved therapeutic approaches that directly promote neuronal remyelination and/or survival. As an approach to developing such therapies, we are working to establish an in vitro myelination assay that works with human cell culture systems, with the goal of performing a high-content screen of small molecule libraries to identify lead molecules that promote myelination, and thereby help to develop a novel strategy to complement current approaches for MS treatment.
We used CRISPR/Cas9 genome editing to generate knock-in reporter stem cell lines for OPCs and oligodendrocytes (OLs). After differentiation the reporter OPCs can be purified using either FACS sorting or immunopurifcation. We have successfully used this strategy to develop reporters for stem cell-derived RGC differentiation and purification. We will then co-culture purified RGCs and purified OPCs to establish an in-vitro myelination system. Either the co-culture or the immunopurified OPCs will be plated into a 384-well or a 1536-well culture format for high-throughput screening. An Echo (Labcyte) acoustic liquid handler will be used to aliquot library compounds, and a tip-based automated robotic liquid handling system will be used to add other reagents and to seed the cells. Imaging will be performed with a Cellomics ArrayScan high-capacity HCS reader.
We successfully differentiated stem cells into OPCs and RGCs and purified them. With our protocol, O4+ OPCs are detected as early as day 60 and myelin basic protein (MBP+) OLs within 100 days of differentiation. Using CRIPSR/Cas9, we have successfully generated a dual knock-In reporter cell line that expresses tdTomato driven by the PDGFRa promoter, which should express GFP when MBP is expressed. We also have a stem cell reporter line for RGCs that expresses tdTomato driven by the RGC specific gene POU4F2/BRN3B. In addition, using our reporter cell lines, we can purify PDGFRa+/tdTomato+ OPCs and tdTomato+/POU4F2+ RGCs with 90% and 95% purity, respectively.
With successful differentiation and purification of stem cell-derived RGC and OPC reporter cells, we are ready to initiate small molecule screening to identify compounds that promote myelination.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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