Abstract Rationale Development of rapid platforms for the in vivo evaluation of patient-specific pathogenic variants is critically important for advancing the understanding of monogenic human diseases. Conventional models are often time-consuming and limited in their ability to capture cell type-specific effects of human mutations during organogenesis. Here, we describe a novel strategy combining blastocyst complementation with single-cell RNA sequencing (scRNA-seq) to assess the functional consequences of evolutionarily conserved human mutations across multiple cellular lineages in mouse embryos. Methods We focused on the S52F FOXF1 heterozygous pathogenic variant identified in patients with Alveolar Capillary Dysplasia (ACD). Mouse embryonic stem cells (ESCs) carrying this FOXF1 variant were generated and injected into blastocysts to create mouse chimeras. Lung development was evaluated by scRNAseq at the single-cell level to determine lineage-specific differentiation defects. To uncover conserved molecular mechanisms, we integrated scRNAseq data from mouse chimeras with publicly available human ACD scRNAseq and single-nucleus ATACseq datasets. Results ESCs harboring the FOXF1 S52F variant exhibited impaired differentiation into endothelial cells, pericytes, and fibroblasts during embryonic lung development. Single-cell transcriptomic analysis revealed disrupted gene expression networks associated with angiogenesis and mesenchymal maturation. Comparative analysis demonstrated parallels between the dysregulated pathways in mouse chimeras and those observed in human ACD datasets, highlighting conserved pathogenic mechanisms driven by FOXF1 dysfunction. Conclusions We established a proof-of-principle that blastocyst complementation combined with scRNA-seq provides a fast and robust in vivo platform to evaluate pathogenic human variants. This approach enables the identification of molecular mechanisms underlying monogenic diseases such as ACD, offering a powerful tool for functional genomics and precision medicine. This abstract is funded by: NONE
Wen et al. (Fri,) studied this question.