Abstract
Purpose :
Promoting remyelination of neurons in the central nervous system is a promising approach for treatment of optic neuritis secondary to multiple sclerosis (MS) and other demyelinating neurodegenerations, and also may be important in the development of strategies for optic nerve regeneration. Current screens for drugs that promote myelin production predominantly utilize rodent cells. Given the important differences in mouse and human oligodendrocyte (OL) development, the goal of this project was to develop a human stem cell-derived OL precursor cell (OPC)-based platform for high-throughput screening of potential myelination promoting compounds.
Methods :
Using CRISPR/Cas9-based genome editing, an identification and purification (IAP) tag was knocked-in just prior to the translational stop site of the endogenous PDGFRa, a marker gene for OPCs. The IAP tag consists of sequences for tdTomato and a unique mouse cell-surface protein, Thy1.2, separated by a 2A peptide. In this IAP system, upon PDGFRa expression, tdTomato localizes to the cytoplasm whereas Thy1.2 localizes to the cell surface, allowing the PDGFRa expressing OPCs to be immunopurified via Thy1.2 microbeads. We also knocked-in GFP and secreted Nano luciferase (secNluc) reporter proteins such that they are driven by the OL maturation and myelination markers PLP1 and MBP respectively. Since the secNanoLuc is separated from the MBP gene product by a 2A sequence, NanoLuc is secreted into the culture media when MBP is expressed. This allows MBP expression to be quantitated using a small aliquot of the cell culture media. In addition, since the GFP expression is representative of PLP1 expression, image-based high-content screening (HCS) can also be performed with these cells.
Results :
We have screened several libraries of bioactive molecules, and identified 23 compounds that enhanced NanoLuc activity greater than five standard deviations above the mean. Several of the compounds we identified, including muscarinic receptor antagonists, Cytochrome P450 inhibitors and SERMs were recently reported as re-myelination compounds in a rodent model system. Additionally, several molecules and potential targets that have not been previously implicated in OL differentiation and function were also identified.
Conclusions :
We developed a human OPC-based drug screening platform for the discovery of remyelinating compounds.
This is a 2020 ARVO Annual Meeting abstract.