Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury

Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this, immunocompetent adult female rats received a controlled cortical impact injury (CCI) or sham surgery. LMNSC008 cells or a vehicle (VEH) were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. The data demonstrate a robust proof-of-concept for LMNSC008 therapy for TBI and provides a strong rationale for IN delivery for translation in TBI patients. NanoString RNAseq sample preparation and data acquisition Brain tissue was dissected and RNA was isolated from snap frozen tissues according to a diagram in figure S6 and half was used for Nanostring analysis and half for protein ELISA assays. RNA expression in the ipsilateral and contralateral hemispheres of NSC and VEH-treated TBI brains was analyzed using the NanoString nCounter platform (NanoString Technologies) by digitally detecting and counting RNA expression in a single reaction without amplification. Each assay included six positive and eight negative RNA assay controls, plus ten mRNA housekeeping controls. RNA was hybridized with the Codeset from the gene panel at 65°C for 16 h. The post-hybridization probe-target mixture was quantified using the nCounter Digital Analyzer, and all data analyses were performed on nSolver (NanoString Technologies). All raw data were first normalized with internal positive and negative controls to eliminate variability unrelated to the samples, then normalized to the selected housekeeping genes using Geometric Means methods. Statistically significant (p<0.05) and < 2 fold up- or down- regulated genes were analyzed and clustered as described below.