Engineering Personalized Neural Tissue Using Cell Permeable Transcription Factors

Introduction: The transcription factor Ascl1 plays a key role in neural development and previous work by the Wernig group demonstrated that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly in neurons. Here we report on a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) that can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. As hiPSCs can be generated from a patient's own cells, this method could potentially enable engineering of patient specific neural tissue.

Results: We evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media, which was 15 µg/mL. The protein did have concentration dependent effects on hiPSC differentiation into neurons. We then determined how often this protein needed to be added to the cell culture media to induce neuronal differentiation, selecting the following time points for media changes: 12, 24, 48, and 72 hours. These media changes were performed over 8 days and then the cells were stained for the presence of Ascl1-IPTD and the early neuronal marker Tuj1. All groups showed positive staining for the Ascl1 protein and exhibited high levels of Tuj staining, indicating that the frequency of media changes did not have an effect. Having demonstrated the effectiveness of the Ascl1-IPTD protein at differentiating hiPSCs into immature neurons, we then compared its ability to generate more mature neurons to a standard, commercially available protocol for generating neural progenitors and negative control cultures over a 12 day culture period. Both the cultures treated with Ascl1-IPTD and the positive control exhibited high levels of NeuN staining, indicating differentiation into mature neurons. Using Neurotrack software, we were able to analyze the average neurite length and amount of neurite branching observed for these cultures. The cells treated with Ascl1-IPTD had an average neurite length of 3.0372 mm/mm² compared to 2.1649 mm/mm² for the positive control. Likewise, the average number of branch points for the cells treated with Ascl1-IPTD was 28.052 1/mm compared to 16.743 1/mm for the positive control. The negative control showed little neurite extension and branching.

Discussion: While transcription factors play important roles in stem cell maintenance and differentiation, they are not often used to influence cell behavior due to their instability. The main goal of this work was to determine if using intracellular protein delivery technology could turn the transcription factor Ascl1 into a effective neuronal differentiator of stem cells, which it did. Our novel Ascl1-IPTD construct is both stable in the media and has a potent effect on stem cell behavior, making it an attractive alternative to viral based methods of increasing Ascl1 expression in cells. This study demonstrates how the Ascl1-IPTD protein can be used to efficiently and inexpensively derive neurons from hiPSCs compared to traditional protocols. Currently, we are determining if this Ascl1-IPTD protein can be used to directly reprogram human fibroblasts into neurons.