Motor neuron differentiation from pluripotent stem cells and other intermediate proliferative precursors that can be discriminated by lineage specific reporters.

We have used a four stage protocol to generate spinal motor neurons (MNs) from human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs). These stages include the pluripotent stem cell (PSC) stage, neural stem cell (NSC) stage, OLIG2 expressing motor neuron precursor (MNP) stage, and HB9 expressing mature-MN stage. To optimize the differentiation protocol reporter lines marking the NSC and MNP stages were used. The NSC stage is a pro-proliferative precursor stage at which cells can be directed to differentiate to other neural types like cortical neurons also, in addition to MNs; thus, NSCs can be expanded and stored for future differentiation to different neural types thereby, shortening the differentiation interval as compared to the complete process of differentiation from ESCs or iPSCs. Additionally, we find that OLIG2 positive cells at the MNP stage can be cryopreserved and then recovered to continue the process of MN differentiation, thereby providing a highly stable and reproducible technique for bulk differentiation. MNPs were differentiated to MNs expressing the marker HB9 demonstrating that mature-MNs can be generated with this protocol.