F. Takaesu
Johns Hopkins University School of MedicineAuthor Of 1 Presentation
P0775 - Development of a screening platform that uses genome engineered hPSC-Derived OPCs for the discovery of remyelination promoting compounds (ID 1677)
Abstract
Background
Promoting remyelination of neurons in the central nervous system (CNS) is a promising approach for treatment of multiple sclerosis (MS) and other demyelinating neurodegenerations. Currently, the systems being used to screen for such remyelinating drugs predominantly use rodent cells. Given the important differences in mouse and human oligodendrocyte (OL) development and gene expression, validating and expanding the mouse studies in a human system is important. In addition, drug discovery performed with human cells has a better probability to identify leads that will translate into effective treatments.
Objectives
The goal of this project was to develop a human stem cell-derived oligodendrocyte precursor cell (OPC)-based platform for high-throughput as well as high-content screening (HTS and HTC) of potential myelination promoting compounds.
Methods
We used CRISPR/Cas9-based genome editing to modify three OPC and OL specific genes in a hESC line (WA09). First, an identification and purification (IAP) tag was knocked-in just prior to the translational stop site of the endogenous PDGFRα, a marker gene for OPCs. The IAP tag consists of tdTomato and a unique mouse cell-surface protein, Thy1.2, separated by a 2A peptide. In this IAP system, upon PDGFRα expression, tdTomato localizes to the cytoplasm whereas Thy1.2 localizes to the cell surface, allowing the PDGFRα 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 the NanoLuc activity, which represents the 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 a number of compounds that promote maturation of human OLs. As a validation of our assay system, several of the compounds we identified, including muscarinic receptor antagonists, Cytochrome P450 inhibitors, SERMs and ROCK inhibitors were recently reported as myelination promoting compounds in a rodent OPC in vitro 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. HTS performed using this platform identified a number of lead molecules that promote OL maturation, and could enhance myelination.