Grant Award Details
- To define the transcriptomic responses of human embryonic stem cell-derived microglia to repeat mild closed head injury and validate the spatial and temporal gene expression patterns by CRISPR editing microglia to deliver a reporter peptide to injury sites within the brain at multiple post-injury timepoints.
Grant Application Details
- Mapping the spatial and temporal responses of hESC-derived microglia to repeat mild closed head injury to identify therapeutic targets and mechanisms
We will generate an RNA activation map of human stem cell derived microglia activation states following brain injury to then test a new gene-edited microglia peptide delivery mechanism.
Bottlenecks with the time and sex-dependent human microglia responses to repeat mild closed head injury and questions surrounding the delivery and efficacy of a trophic factor as a therapeutic.
Major Proposed Activities
- Generate an RNA map of the human microglial response to repeat mild concussions at multiple post-injury timepoints on mice transplanted with microglia derived from male and female stem cells.
- Use microscopy to histologically validate the microglia populations identified in Activity 1 express candidate genes from the early vs late and/or male vs female injury responses.
- CRISPR edit male and female stem cell lines to append the sequence for a secreted trophic factor coupled to a reporter peptide at the end of the selected injury-response genes found in Activity 1.
- Verify that the microglia generated from CRISPR editing are different than the parental lines as designed in Activity 3 (quality control).
- Stimulate the CRISPR edited microglial lines in cell culture to confirm that the trophic factor/reporter peptide responds to injury and is secreted.
- Perform repeat mild concussions on mice transplanted with the CRISPR engineered microglial cell lines and verify that the inserted trophic factor/reporter peptide is delivered to the injury site.
This research could benefit California by discovering genes that are specifically upregulated in microglia in response to brain injury. Editing a gene to deliver a trophic factor to the injury could be a pathway for treating concussion/closed head injuries or even more severe traumatic brain injuries (which effect >230,000 Californians/year and cost CA ~9.6 billion every year). This bottleneck could also open the door for stem cell based treatments of a variety of neurological conditions.