Proliferation history and transcription factor levels drive direct conversion

Abstract

AbstractThe sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential forin vivotherapies.One Sentence SummaryUsing a systems and synthetic biology approach to study the molecular determinants of reprogramming, we find that proliferation history and transcription factor levels drive cell fate in direct conversion to motor neurons.Highlightsminimal high-efficiency cocktail allows systematic interrogation of the reprogramming processhistory provides a principal axis to distinguish transcription factors’ influenceof the transcription factor cocktail impacts reprogramming efficiency and dynamicsof individual transcription factors differentially influence the rate of reprogrammingearly hyperproliferation increases direct conversion of adult human fibroblastsptimal cocktail allows neurotrophic factor-free reprogramming andin vivografting