California's Stem Cell Agency

Research Objective

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.

Impact

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.