Molecular Approaches to Increase Stroke Resiliency Using Human Pluripotent Stem Cell- Derived Neurons

Elise MacDonald

Document Type

Article

Publication Date

Summer 2022

Keywords

JMG

JAX Location

In: Student Reports, Summer 2022, The Jackson Laboratory

Abstract

Strokes, which are caused by an interruption in blood supply to the brain, damage neurons and can lead severe long-term ailments such as paralysis. Genetic diversity contributes to a range of outcomes after stroke. By analyzing the transcriptomes of mouse pluripotent stem cell (mPSC)- derived neurons and human pluripotent stem cell (hPSC)-derived neurons subject to in vitro stroke conditions, we can identify key regulatory factors contributing to stroke resilience. The Pera lab has previously identified three transcription factors (TFs), IRF7, VDR, and TFAP2C, that are significantly upregulated across stroke-resilient cell lines. Using small molecule agonists/antagonists of these TFs and CRISPR activation (CRISPRa), it is possible to modify hPSC-derived neurons to increase their resilience to stroke, thus offering insights into personalized and novel therapeutic approaches. Preliminary analysis suggests that combinations of small molecules, such as EB1089 + Selenite + R08191/IFNARIN are the most effective in increasing stroke resiliency in hiPSC derived neurons. We attempted to establish a hiPSC line that stably expresses dCas9-VPR after differentiation into neurons. Although the vast majority of cell clones dies, we successfully differentiated a clone into cortical neurons and characterized the effectiveness of the technology to integrate into the genome. It is evident that the dCas9-VPR transgene inserts randomly into the genome since the success rate of transgenic hiPSC to neuron differentiation is quite low. It would be beneficial to continue to study small molecule treatments across various hiPSC-derived cortical neuron lines, as well as dCas-9VPR technology that transfects into a safe harbor locus in the genome to ensure expression.

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