Higher throughput workflow with sensitive, reliable and automatic quantification of myelination in vitro suitable for drug screening

Abstract

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC–OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy.