(893) Induced Pluripotent Stem Cell-Derived Monocyte Response to Risk-Stratified Pediatric Septic Serum

Introduction: Heterogeneity in pediatric septic shock has limited the development of novel therapeutics to improve morbidity and mortality. The Pediatric Sepsis Biomarker Risk Model (PERSEVERE) II is validated to estimate baseline mortality risk in children with septic shock, but the contribution of risk-stratified serum to the host response remains unclear. Human induced pluripotent stem cells (iPSCs) provide a renewable source of monocytes for study. We hypothesized that exposure of iPSC-derived monocytes to risk-stratified serum would identify different patterns of gene expression.

Methods: Human iPSCs were differentiated into monocytes. Flow cytometry was performed to verify CD14 expression. iPSC-monocyte function was evaluated with lipopolysaccharide (LPS) stimulation (1 ug/mL) for 24 hours followed by RNA collection. Gene expression was evaluated with the Nanostring nCounter system using the Human Inflammation Panel v2 (250 genes tested) and analyzed using the ROSALIND platform. iPSC-monocytes were exposed to 10% PERSEVERE risk-stratified serum pooled from children with septic shock in media for 24 hours followed by RNA collection. Differentially expressed genes (DEGs) between iPSC-monocytes exposed to high- and low-risk serum and controls were evaluated. iPSC-monocytes underwent CD14-based immunomagnetic separation to validate the unselected results, and the cells were subsequently exposed to LPS prior to RNA collection and analysis.

Results: Seventy percent of monocytes were CD14 positive 20 days after differentiation. Sixty-one DEGs (>2-fold change, p-adj < 0.01) were identified between iPSC-monocytes exposed to LPS versus control, including TNF-alpha and chemokine signaling pathways. Exposure to high PERSEVERE serum resulted in 16 DEGs compared to control (1.5-fold change, p-adj < 0.05) and 16 DEGs compared to low-risk serum (>1.5-fold change, p-value < 0.05). After CD14 selection 55 DEGs (>5-fold change, p-adj < 0.01) were identified between LPS exposure and control, including 36 of the same genes identified pre-selection.

Conclusions: Gene expression was comparable between iPSC-monocytes pre- and post-CD14 selection. Exposure to high PERSEVERE-risk serum resulted in different patterns of gene expression compared to low-risk serum and controls, guiding further mechanistic investigation.