An SMA project report: neural cell-based assays derived from human embryonic stem cells

Human embryonic stem (ES) cells are promising resources for developing new treatments for neurodegenerative diseases. Spinal muscular atrophy (SMA) is one of the leading causes of childhood paralysis and infant mortality. SMA is caused by inactivation of the survival motor neuron-1 (SMN1) gene. The nearly identical SMN2 gene contains a silent polymorphism that disrupts splicing and as a result cannot compensate for loss of SMN1. The SMA Project was established by the National Institute of Neurological Disorders and Stroke (NINDS) as a pilot effort to establish a fully transparent coalition between academics, industry, and government to create a centralized network of shared resources and information to identify and test new SMA therapeutics. As one of the funded projects, the work described here tested the feasibility of generating a SMA cell-based assay using neural lineages derived from human ES cells approved for National Institutes of Health (NIH)-funded research. Minigene cassettes were constructed, employing firefly luciferase or green fluorescent protein (GFP) as reporters for splicing efficiency of SMN1 and/or SMN2 under the control of the SMN1, SMN2, or cytomegalovirus (CMV) promoters. Transient transfection of proliferating neuroprogenitors in a 96-well format with plasmid DNA or adenoviral vectors showed differential levels that correlated with the splicing minigene and the promoter used; luciferase activities with SMN1 splicing minigenes were higher than SMN2, and the CMV promoter generated higher levels of activity than the SMN1 and SMN2 promoters. Our results indicate that human ES cell-derived neuroprogenitors provide a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.