Establishing Robust Characterization of Novel Therapeutic Responses and Disease Models of Necrotizing Enterocolitis

PROJECT SUMMARY Necrotizing enterocolitis (NEC) is a life threating intestinal disease in premature infants, with high mortality rates of 20-40%. To date, there are no approved therapies available for NEC. To address this challenge, we have shown that human-derived placental stem cells (hPSC) can ameliorate both the dysregulated inflammation and damaged epithelium in NEC. Based on the potent paracrine signaling mechanism of hPSC, we have identified hPSC extracellular vesicles (hPSC-EV) as a promising cell-free therapy with distinct advantages in translational potential. However, the efficacy of the hPSC-EV therapy compared to hPSC therapy is unclear. Elucidation of both the repaired and unaffected NEC-associated expression profiles following therapy is also needed to fully optimize a hPSC-based therapy approach. With similar pathology to medically-managed NEC in human infants, the rat pup model of NEC is well-suited to identify disease-associated cellular and transcriptional changes in NEC disease and with hPSC-based treatment. Human enteroid model systems are also often employed to investigate intestinal diseases and therapeutics for improved translation to the human disease. However, a NEC enteroid model system has not yet been rigorously validated for the ability to recapitulate NEC disease features of the epithelium. Based on promising preliminary data, we now seek to address these 2 challenges in NEC by defining the efficacy of hPSC-EV therapy, determining the cellular and transcriptional changes in NEC disease and therapy repair, and establish a novel human NEC enteroid monolayer model system. We hypothesize that integrated characterization of in vivo NEC pathogenesis, in vivo hPSC-based therapeutic responses, and an in vitro human NEC model system will elucidate unique cell-specific expression profiles of NEC disease and repair pathways and validate the retention of the NEC epithelial-associated expression profile in a novel in vitro human NEC enteroids-based model system. The aims of this application reflect an integrated study of in vivo and in vitro NEC disease and therapeutics to: (1) assess the efficacy of hPSC-EV as compared to hPSC therapy, (2) identify cell population andl cell-type specific expressional profiles during NEC disease and hPSC-induced repair, and (3) establish human NEC enteroids-derived model systems as a high-throughput model system for human disease translation. In Aim 1, we will determine the efficacy of hPSC-EV therapy and utilize scRNAseq to profile shifts in cell populations and identify novel expression profiles within these cells in both disease and repair. In Aim 2, we will utilize bulk RNAseq to determine the ability of 3D enteroids and 2D monolayers derived from resected human NEC tissue to recapitulate known in vivo NEC expression profiles. Combined the proposed studies will advance our understanding of targetable repair pathways in NEC and provide a human disease model to improve clinical translation. With strong, supportive preliminary data and impactful proposed studies, this study has a high likelihood of establishing success for building a robust translational foundation. These studies will inform our future translational efforts investigating advanced therapeutic strategies in human NEC.