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

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 LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.

Efficient differentiation of human embryonic stem cells toward dopaminergic neurons using recombinant LMX1A factor.

Direct differentiation of dopaminergic (DA) neurons from human pluripotent stem cells (hPSCs) in the absence of gene manipulation is the most desired alternative to clinical treatment of Parkinson disease. Protein transduction-based methods could be efficient, safe approaches to enhance direct differentiation of human embryonic stem cells (hESCs) to DA neurons. In the present study, we compared the differentiation efficiency of DA neurons from hESCs with and without the application of LIM homeobox transcription factor 1 alpha (LMX1A), a master regulatory protein in the development of the midbrain neurons and SHH proteins. The results obtained revealed that the treatment of hESCs with recombinant LMX1A (rLMX1A) protein along with dual SMAD inhibition led to higher expression of LMX1B, LMX1A, FOXA2, PITX3, EN1, and WNT1 effector endogenous genes and two-fold expression of PITX3. Moreover, the highest expression level of PITX3 and TH was observed when rLMX1A was added to the induction medium supplemented with SHH. To our best knowledge, this is the first report demonstrating the application of TAT-LMX1A recombinant protein to enhance hESC differentiation to DA as shown by the expression of DA specific makers. These findings pave the way for enhancing the differentiation of hESCs to DA neurons safely and efficiently without genetic modification.