RNAseq of oculomotor (OMN) and spinal motor neurons (SC) derived from mouse embryonic stem cells and purified by FACS

Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo. Overall design: A total of 24 samples were analyzed here. Of these, 12 samples are oculomotor neurons derived from mouse embryonic stem cells (mESCs) facilitated by Phox2a over-expression. The remaining 12 samples are spinal motor neurons also mESCs-derived. mESCs were cultured and patterned with appropriate morphogens as embriod bodies for 5 days and neurons purified using FACS and collected as bulk for RNAseq or re-plated for characterization. This was performed for 2 mESC lines (Islet1-GFP/NesEPhox2A for OMN and Hb9-GFP for SC differentiations), resulting in the following 4 groups: OMN GFP positive fraction (n=6), OMN GFP negative fraction (n=6), SC GFP positive fraction (n=6) and SC GFP negative fraction (n=6).