Foxa1 disruption enhances human cell integration in human-mouse interspecies chimeras

Abstract

Blastocyst complementation can potentially generate a rodent model with humanized nasopharyngeal epithelium (NE) that supports sustained Epstein-Barr virus (EBV) infection, enabling comprehensive studies of EBV biology in nasopharyngeal carcinoma. However, during this process, the specific gene knockouts required to establish a developmental niche for NE remain unclear. We performed bioinformatics analyses and generated Foxa1 mutant mice to confirm that Foxa1 disruption could potentially create a developmental niche for NE. Subsequently, MYD88-inactivated human pluripotent stem cells (hPSCs) were constructed and complemented with Foxa1-deficient mouse blastocysts, with Nosip-deficient mouse blastocysts as a control. The chimerism of human cells in mouse embryos was evaluated from E8.5 to E12.5 using genomic DNA PCR and immunohistochemistry. Our bioinformatics analysis indicated that the expression patterns of Foxa1 in E8.5 to E16.5 mouse embryos underscore its critical role in NE development. The generated mice with Foxa1 disordered region mutations displayed morphological abnormality in NE, suggesting Foxa1-knockouts could potentially establish a developmental niche for NE. In chimeric assays, human cells integrated into 80.00% of Foxa1-deficient embryos, compared with the 4.17% in controls. Immunohistochemistry results revealed robust proliferation of human cells in Foxa1-deficient mouse embryos. However, chimeras from Foxa1-deficient mouse embryos did not survive beyond E10.5, hindering the evaluation of human cell integration in mouse NE. Foxa1 disruption in mouse embryos significantly enhances the integration of human cells in human-mouse interspecies chimeras, thereby facilitating the generation of endoderm-derived organs through blastocyst complementation. Overcoming chimeras' embryonic lethality is crucial for successfully generating humanized NE in Foxa1-deficient mouse embryos.

Keywords: Foxa1; Blastocyst complementation; Human-mouse interspecies chimerism; Nasopharygeal epithelium.