(317e) A Streamlined Protocol for Highly-Efficient Reprogramming to Induced Motor Neurons

Cellular reprogramming converts common, accessible cells into rarer, inaccessible cell types, such as neurons. We recently developed a chemical-genetic cocktail that increases reprogramming of mouse embryonic fibroblasts (MEFs) to induced motor neurons (iMN) at 100-fold higher rates over the base delivery of six reprogramming transcription factors. This specific cocktail increases reprogramming by increasing the population of hyperproliferating, hypertranscribing cells (HHCs), which reprogram at near-deterministic rates. However, we observed significant variance in the reprogramming efficiency across batches of cells, which may be due to variability in media conditions as well as the differential delivery of six different viruses which each encode a transcription factor. To improve this process, we aimed to simplify the reprogramming process by developing a streamlined protocol for highly efficient reprogramming. Specifically, we sought to reduce the number of viruses required for co-delivery of reprogramming factors and the high-efficiency genetic cocktail. Additionally, we explored eliminating costly and potentially variable protein supplements during reprogramming.

To achieve this goal, we identified the minimal set of transcription factors sufficient to induce robust MEF-to-iMN reprogramming: Ngn2, Isl1, and Lhx3 (NIL). Interestingly, we find that by reducing the number of transcriptions factors used from the original six to just three (NIL), we massively increase the population of HHCs and reprogramming yield, increasing the yield of iMNs another 20-fold. We explored optimizing the transcription factor stoichiometry by rearranging the order of the transcription factor cassette. We found that varying stoichiometries reprogramming factors (NIL) have minimal effects on the yield from our highly efficient reprogramming protocol. Our results suggest that total levels rather than stoichiometric expression of reprogramming factors dictate iMN reprogramming yields. In addition, to simplify our protocol and remove extra sources of variability, we explored reprogramming in the absence of neurotrophic factors. We find that neurotrophic factors are not required for robust iMN reprogramming in our high-efficiency system, minimizing both cost and complexity of the process.

Taken together, this work outlines a highly efficient and robust iMN reprogramming protocol that is consistent with our previous finding that expanding the population of highly-plastic cells in reprogramming increases reprogramming yields. With this massive increase in reprogramming yield, we can readily obtain sufficient number of cells to perform biochemical assays, allowing us to precisely map the molecular mechanisms of cell-fate transitions during high-efficiency reprogramming.