Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

Abstract

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of <i>LMX1A, FOXA2</i> and <i>PITX3</i> is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that <i>LMX1A</i> significantly increases <i>TH</i>-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while <i>FOXA2</i> and <i>PITX3</i> increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.