Reparative Mechanisms of STEM Cell-Based Therapy Using Extracellular Vesicles Derived from Human Placental STEM Cells for Necrotizing Enterocolitis

Necrotizing Enterocolitis (NEC) is a life-threatening gastrointestinal disease that primarily affects premature infants born at 34 weeks or less. It is characterized by the infection, inflammation, and death of intestinal tissues in the ileum of the small intestine. Though the etiology and pathogenesis of NEC remain unknown, several factors such as ingestion of formula instead of breast milk, hypoxic injury, and bacterial invasion, have been shown to contribute to the development of this disease. To date, therapy options have been limited to surgical and medical management, often leading to subsequent issues such as short bowel syndrome in the infant’s future.

Recent cell therapy studies show that using the paracrine signaling mechanism, human placental derived stem cells (hPSC) therapy promotes epithelial healing of NEC intestinal damage. Although this has proven to be an effective therapeutic, the use of Extracellular Vesicles (EVs) produced from hPSCs provides a novel cell-free therapeutic approach for optimized clinical translation. In this study, we tested the efficacy of hPSC-EVs in ameliorating the NEC disease damage to proliferation, progenitor stem cells, LGR5+ active intestinal stem cells, and differentiated epithelial cells.

We used breastfed (BF), diseased (NEC), and treated (NEC-EV) ileal tissues from newborn Sprague-Dawley rat pups for our investigation. We utilized cell-specific markers through immunofluorescence staining to quantify changes in cell number using the GNU Image Manipulation Program (GIMP) and a newly developed ImageJ plug-in. We hypothesized that EV therapy will promote the inherent mechanism of the small intestine to repair epithelial damage from NEC disease, specifically within particular cell types. We quantified the following markers: Ki67 (proliferation), SOX9 (intestinal progenitor/stem cells), OLFM4 (LGR5+ intestinal stem cells), and Ezrin (enterocytes). BF, NEC, and NEC-EV ileums were immunostained with specific antibodies for each marker. The number and location of positively stained cells were quantified. From this analysis, we were able to identify changes within proliferation, progenitor stem cells, LGR5+ active intestinal stem cells, and differentiated cell types in the NEC and NEC-EV tissues. Additionally, we were able to quantify the distribution of each marker along the crypt-villus axis.

Based on our findings and past studies, we observed that EVs derived from human placental stem cells are an effective therapeutic for Necrotizing Enterocolitis. Future studies will focus on the additional immune modulation benefits of this EV therapy, providing a better understanding of the mechanisms of action for this therapeutic approach and guiding future translation in the NICU.