Integrated analyses of in vivo mouse models and liver organoids reveal MFG-E8-mediated protection against hepatocyte injury.

Milk fat globule-EGF factor 8 (MFG-E8) is a multifunctional glycoprotein implicated in tissue homeostasis and repair. Although its roles in intestinal and pancreatic injury are well established, its function in hepatic regeneration remains poorly defined. In this study, we investigated MFG-E8 expression and activity using a hepatocyte-specific apoptosis model (3xTg-iHAP) and liver organoid cultures from wild-type (B6WT) and Mfge8 knockout (Mfge8-/-) mice. In doxycycline (Dox)-treated 3xTg-iHAP mice, Mfge8 mRNA and MFG-E8 protein were markedly upregulated, peaking at 48 h after hepatocyte injury. RNAScope-Immunostain Integrated Codetection Workflow (ICW) demonstrated that under basal conditions, Mfge8 expression was localized to cholangiocytes, but following injury, hepatocytes exhibited robust transcriptional induction. In vitro, three-dimensional (3-D) organoids showed no overt genotype-specific differences between B6WT and Mfge8-/- mice; however, two-dimensional (2-D) monolayers derived from Mfge8-/- mice exhibited cytopathic disintegration by day 5. This phenotype was partially rescued by recombinant MFG-E8 (rMFG-E8). Transcriptomic analysis by RNA sequencing (RNA-seq) confirmed loss of Mfge8 expression in knockout cultures, accompanied by an approximately eight fold increase in Hddc3, a ferroptosis-associated gene, suggesting a potential mechanism underlying structural instability ex vivo. These findings demonstrate that MFG-E8 is indispensable for maintaining hepatic epithelial integrity. Loss of MFG-E8 sensitizes hepatocytes to ferroptosis stress, providing mechanistic insight into impaired liver repair and highlighting MFG-E8 as a potential therapeutic target to enhance hepatic regeneration.NEW & NOTEWORTHY This study uncovers a novel role of milk fat globule-EGF factor 8 (MFG-E8) in liver regeneration. Hepatocytes upregulate Mfge8 after injury, shifting from its basal expression in cholangiocytes to hepatocytes. Loss of MFG-E8 destabilizes hepatocyte monolayers, a defect partially rescued by recombinant MFG-E8 (rMFG-E8). Transcriptomic analysis revealed Hddc3 upregulation in Mfge8-/- mice, linking MFG-E8 deficiency to ferroptosis. These findings identify MFG-E8 as a key regulator of hepatic epithelial integrity and a potential therapeutic target for liver repair.