PROJECT SUMMARY Significant neurological delay is emerging as one the most important current challenges for children with congenital heart disease (CHD), yet few treatment options are currently available. In our first period of funding, we proposed the use of cardiopulmonary bypass (CPB) as a cell delivery system in infants with CHD as a novel approach for improving the neurological impairments in CHD. Our published and unpublished work has demonstrated the efficacy and utility of this approach, determining the systemic effects of delivery bone marrow- derived mesenchymal stromal cell (BM-MSC) via CPB and the effect on white matter (WM) and sub-ventricular zone (SVZ) development. Most notably, the first award successfully led to development of a phase 1 clinical trial termed “MeDCaP” at Children’s National. Using our translational piglet model, we have demonstrated cellular, structural, and behavioral improvements after BM-MSC delivery through CPB and generated critical information for bench-to-bedside translation. However, the mechanisms underlying the therapeutic action of BM-MSCs still remain largely unknown. This R01 renewal will address the key knowledge gaps with the goal of further enhancing our cell-based treatment for neuroprotection in the CHD population. Exosome is a class of extracellular vesicles loaded with bioactive molecules such as microRNA (miRNA). Exosomes derived from BM- MSCs (BM-MSCexo) can play a major role in the effects on surrounding cells and tissues and elicit favorable responses in various diseases. We have established a pipeline for post-cell delivery integrated transcriptomic analysis of exosomal miRNAs from BM-MSCs and host tissue mRNA. Our preliminary studies have identified the BM-MSCexo-derived miRNAs as putative key drivers of reduced neuronal apoptosis and microglial activation observed after BM-MSC treatment in the cerebral cortex. The overarching goal of this renewal proposal is to establish detailed molecular signatures from critical cell populations for tissue repair and regeneration at single cell resolution after BM-MSC delivery; we will then use those molecular signatures as roadmaps to identify novel molecular entities within the BM-MSCexo that account for the disease-modifying bioactivity in tissue injury after pediatric cardiac surgery. The renewal studies will test our central hypothesis that specific exosomal cargo constituents from BM-MSCs promote repair and regenerative processes both through neural progenitors and regulatory T cells, thereby improving neurological outcomes and post-operative course. To retroactively identify key exosomal bioactive molecules, we will determine the transcriptional and chromatin landscape of three specific cell populations: 1) SVZ neural stem and progenitor cells; 2) WM oligodendrocytes; and 3) regulatory T cells. Together with our ongoing clinical trial established based on the previous award, identifying molecular signatures of BM-MSC treatment and mining specifi...