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 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.
Highlights
The human nervous system contains at least hundreds of distinct subtypes of neurons that are necessary to create complex circuits
The mRNA template was constructed with a T7 promoter and was engineered with untranslated regions (UTRs) of highly stable Human b–globin (Hbb) at both 5’ and 3’ ends (Angel and Yanik, 2010), since UTRs play a significant role in mRNA stability (Yu and Russell, 2001; Jiang et al, 2006)
In addition to growth factor-based differentiation protocols, exogenous overexpression of transcription factors using DNA vectors can partially enhance the number of tyrosine hydroxylase (TH) positive dopaminergic neurons during differentiation of pluripotent stem cells under various culture conditions
Summary
The human nervous system contains at least hundreds of distinct subtypes of neurons that are necessary to create complex circuits. Generation of these specific neuronal cell types from progenitor cell populations in vitro is important for basic science, drug screening, and potentially for cell-based therapeutics. Significant effort has shown that known extracellular trophic factor cocktails, useful, do not enable sufficient control and specificity to efficiently reprogram progenitor populations to a desired differentiated state (Kriks et al, 2011; Maroof et al, 2013; Shi et al, 2012a; Shi et al, 2012b). Neurons inherit the same genes, cell fate decisions during neurogenesis are mediated through the unique and highly coordinated temporal and spatial expression of hundreds of transcription factors.
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