689 - Attenuation of Inflammation and Restoration of Neurogenesis in the Brain of Fetuses with Congenital Diaphragmatic Hernia Treated with Stem-Cell Derived Extracellular Vesicles

Publication Number: 2672.689

Emily Lo, The Hospital for Sick Children, Toronto, ON, Canada; Fabian Doktor, The Hospital for Sick Children, Toronto, ON, Canada; Victoria Fortuna, University of Toronto Temerty Faculty of Medicine, Toronto, ON, Canada; Min Yi Feng, The Hospital for Sick Children, Toronto, ON, Canada; Giuseppe Speziale, The Hospital for Sick Children, Toronto, ON, Canada; Brian T. Kalish, Boston Children's Hospital, Boston, MA, United States; Augusto Zani, Washington University, St. Louis, MO, United States

Background: Neurodevelopmental impairment (NDI) in CDH infants has been traditionally attributed to postnatal factors, including surgery and extracorporeal life support. Yet emerging evidence from antenatal imaging studies suggests that brain injury may be sustained in utero. While there is currently no established treatment to prevent brain injury in children born with CDH, antenatal administration of stem cell-derived extracellular vesicles (EVs) have demonstrated efficacy in improving lung development in CDH models. Thus, EVs may also be a therapeutic strategy in rescuing brain injury as they can cross the blood brain barrier.

Objective: To investigate the difference in gene expression in major cell types in the brain of fetal mice with left CDH (LCDH) and the therapeutic potential of amniotic stem cells-extracellular vesicles (AFSC-EVs) in CDH-related brain injury.

Design/Methods: Experiments were approved under ethical protocol (IACUC#64247) (Figure 1). The brains from the three groups (control, LCDH, and LCDH+EVs) were harvested for highly multiplexed in situ spatial transcriptomic analysis to detect region- and cell-type-specific gene expression. The cortical plate of the cortex was selected for analysis with Seurat v5 package running R (v4.4.0) (Figure 2). CellTypist was used to semi-automatically annotate cells and to determine the major cell types in the fetal mouse brain. Differential gene expression was compared between the groups using MAST while adjusting for biological replicates as covariates. Benjamini-Hochberg adjusted p-value < 0.05 was considered significant. Gene Ontology was used to interpret biological functions in selected genes.

Results: Differential gene expression analysis revealed significant increase of Notch2 and Sema4d, markers of inflammation, and Slc2a1, Eng, Angpt2, Vegfra, and Kdr, markers of hypoxia, in axon tract-associated microglia in the LCDH group compared to control (p < 0.05). Treatment with AFSC-EVs significantly decreased Notch2 and Sema4d compared to the LCDH group (p < 0.05) (Figure 3). Furthermore, there was a significant decrease of Cd24a, Mki67, Ascl1, and Sox2 (p < 0.05), markers of neurogenesis and proliferation, in the glutamatergic neuroblasts of LCDH group compared to control. Cd24a was statistically increased following AFSC-EVs treatment.

Conclusion(s): This study shows reduced neural proliferation in the LCDH group, suggesting that LCDH is associated with impaired and suppressed neuronal development. Moreover, as the expression of inflammatory markers in the cortex of LCDH group was attenuated with EVs, EVs may have a protective role in CDH-related brain injury.

Figure 1.
Experimental outline in MacGreen.

Figure 2.
Cortical plate of the cortex was selected for analysis in coronal sections of fetal mice brains.

Figure 3.
(A) Volcano plot showing differentially expressed genes between control, LCDH and LCDH+EV groups. Notch2 and Sema4d were among the most significantly upregulated genes in LCDH group. (B) Volcano plot showing differentially expressed genes between control, LCDH, and LCDH+EV group. Cd24a were among the most significantly upregulated genes in control and LCDH+EV group.