Combination of Blastocyst Complementation and Omics Allows Fast Evaluation of Human Pathogenic Mutations in Mice

Abstract Rationale Development of in vivo animal models carrying patient-derived pathogenic gene variants is critical for dissecting molecular mechanisms critical for human lung diseases. However, generation of new murine lines is costly and time-consuming, often requiring years before mechanistic studies can begin. Objective To establish a rapid experimental platform using a combination of blastocyst complementation and single-cell RNA sequencing (scRNA-seq) to evaluate the effects of conserved human pathogenic variants on lung development. Methods Heterozygous S52F variant of FOXF1, identified in patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV), was introduced into mouse embryonic stem cells (ESCs). Differentially labeled mutant and wild type (WT) ESCs were injected into blastocysts to generate mouse chimeric embryos. Differentiation of FOXF1+/S52F and WT ESC-derived pulmonary cells was compared using immunostaining, flow cytometry and scRNA-seq. Integration of scRNA-seq datasets from mouse chimera and human ACDMPV lungs was performed to identify conserved molecular mechanisms. Measurements and Main Results FOXF1+/S52F ESCs exhibited the loss of main subtypes among endothelial and mesenchymal cells during lung development. Transcriptomic profiling revealed dysregulated lineage-specific gene expression signatures similar to those observed in human ACDMPV lungs. Cross-species scRNA-seq comparisons uncovered conserved pathways, receptor-ligand interactions and novel downstream FOXF1 target genes, including Igf2, Icam2, Tiam1, Ets1, Lamb1 and Igfbp3. Conclusions Blastocyst complementation combined with cross-species scRNA-seq provides a rapid and efficient approach to identify conserved molecular mechanisms disrupted by human pathogenic variants. Chimeric mouse embryos can serve as a powerful platform for uncovering new molecular mechanisms critical for lung development.