Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion [re-analysis]

Direct conversion generates patient-specific, disease-relevant cell types such as neurons that are rare, limited, or difficult to isolate from common and easily accessible cells such as skin cells. However, low rates of direct conversion and complex protocols limit scalability and thus the potential of cell-fate conversion for biomedical applications. Systems-level optimization of conversion protocols across molecular and cellular scales can support scalable cell manufacturing for diverse application. We use insights from our work on how proliferation and transcription factor levels act together to drive direct conversion to optimize the conversion protocol by examining process parameters including transcript design, delivery via AAV, retrovirus and lentivirus, cell seeding density, and the impact of media conditions. Thus, we report a compact, portable conversion process that boosts proliferation and increases direct conversion of mouse fibroblasts to induced motor neurons to achieve high conversion rates of above 1,000%, corresponding to more than 10 motor neurons yielded per cell seeded which we achieve through expansion. Our optimized, direct conversion process generates functional motor neurons at scales relevant for cell therapies (>10^7 cells) that graft with the mouse central nervous system. High-efficiency, compact, direct conversion systems will support scaling to patient-specific, neural cell therapies. Transcriptional profiling of cells generated by direct conversion of mouse embryonic fibroblasts to induced motor neurons (iMNs) at the terminal stage at 14 dpi. Samples include Hb9::GFP+ sorted iMNs generated with 6FDDRR (6F with p53DD, hRASG12V, RepSox) as well as MEFs and embryonic motor neurons (embMN) for reference populations. The 6F are Ascl1, Brn2, Myt1l, Ngn2, Isl1, Lhx3. *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************