Abstract
Cellular transdifferentiation changes mature cells from one phenotype into another by altering their gene expression patterns. Manipulating expression of transcription factors, proteins that bind to DNA promoter regions, regulates the levels of key developmental genes. Viral delivery of transcription factors can efficiently reprogram somatic cells, but this method possesses undesirable side effects, including mutations leading to oncogenesis. Using protein transduction domains (PTDs) fused to transcription factors to deliver exogenous transcription factors serves as an alternative strategy that avoids the issues associated with DNA integration into the host genome. However, lysosomal degradation and inefficient nuclear localization pose significant barriers when performing PTD-mediated reprogramming. Here, we investigate a novel PTD by placing a secretion signal sequence next to a cleavage inhibition sequence at the end of the target transcription factor–achaete scute homolog 1 (ASCL1), a powerful regulator of neurogenesis, resulting in superior stability and nuclear localization. A fusion protein consisting of the amino acid sequence of ASCL1 transcription factor with this novel PTD added can transdifferentiate cerebral cortex astrocytes into neurons. Additionally, we show that the synergistic action of certain small molecules improves the efficiency of the transdifferentiation process. This study serves as the first step toward developing a clinically relevant in vivo transdifferentiation strategy for converting astrocytes into neurons.
Highlights
Transdifferentiation converts mature cells from one specialized cell type into another by varying gene expression patterns (Tanabe et al 2015)
We adopted the strategy to first lower this threshold by repressing astrocytic gene expression using small molecules to inhibit transforming growth factor β1 (TGF-β1)/SMAD and bone morphogenic protein (BMP)/SMAD signaling pathways, which are jointly responsible for astrocyte commitment and maintenance (Stipursky and Gomes, 2007; Chambers et al, 2009; Yang et al, 2013; Zhang et al, 2015)
Human cerebral cortex astrocytes were primed for 2 days followed by screening of a set of small molecules known to have neuronal transdifferentiation potential: CHIR99021, an inhibitor of glycogen synthase kinase 3 (GSK3) signaling; DAPT, an inhibitor of Notch signaling; forskolin, an activator of cyclic adenosine monophosphate (cAMP) signaling; and isoxazole 9 (ISX9), an activator of calcium-mediated signaling (Zhang et al, 2015; Gascón et al, 2016, 2017; Gao et al, 2017)
Summary
Transdifferentiation converts mature cells from one specialized cell type into another by varying gene expression patterns (Tanabe et al 2015) These expression patterns are controlled by transcription factors, regulatory proteins which bind to DNA promoter regions to activate transcription of key developmental genes. Ectopic expression of these transcription factors in somatic cells can change cell fates without the need for inducing a pluripotent. Neural Transdifferentiation by Novel ASCL1-IPTD state (Tanabe et al, 2015) This process avoids the risk of tumorigenesis associated with transplanting pluripotent stem cell-derived therapies in vivo, where incomplete differentiation leads to uncontrolled proliferation (Gordeeva and Khaydukov, 2017). Performing in vivo transdifferentiation would eliminate the need for cell transplantation and immunosuppression depending on the target application
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