CDX2 Promotes Hepatic Specification of hiPSC-derived Endoderm Through the PI3K–Akt–GSK3β Pathway for Improved Therapeutic Efficacy

Background & Aims: Human induced pluripotent stem cell (hiPSC)-derived hepatic endoderm (HE) is a potential treatment for liver diseases, but its heterogeneity poses challenges. Hepatic specification, the conversion of hiPSC-derived endoderm (DE) into HE, is critical for HE induction. Caudal homeobox transcription factor 2 (CDX2) regulates multiple signalling pathways during embryonic development and directs organogenesis. While CDX2 is a key regulator of organ development, its role during hepatic specification remains unclear. Methods: HE markers were detected using western blotting (WB) and immunofluorescence (IF). The CDX2 regulatory axis was identified using mRNA-seq and bioinformatics analysis. During hepatic specification, the relative pathways PI3K–AKT, WNT, epithelial‒mesenchymal transition (EMT) and extracellular matrix (ECM) were confirmed using WB. Hepatocyte-like functions were evaluated based on glycogen content, indocyanine green (ICG) uptake and albumin levels. The therapeutic efficacy of HE cells was evaluated in a mouse liver injury model. Results: Overexpression of CDX2 resulted in enhanced expression and function of HE markers during hepatic specification. mRNA-seq and bioinformatics analysis revealed differentially expressed genes following CDX2 overexpression that target the PI3K–AKT and WNT pathways, which was confirmed by WB. Furthermore, the results showed that EMT and the ECM degradation were suppressed by CDX2 overexpression (p < 0.05). In addition, SB-3CT, an matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) inhibitor, effectively promoted the formation of HE cells derived from hiPSC-DEs. Functionally, hepatocyte-like cells derived from HE cells through CDX2 overexpression presented increased glycogen levels, ICG uptake and albumin production. On the other hand, liver functions and acute injuries were significantly ameliorated when CDX2-modulated HE cells were transplanted into a mouse model. Conclusions: CDX2 promotes HE formation through inhibition of the PI3K–Akt and Wnt/β-catenin pathways, as well as suppression of EMT and ECM degradation. Moreover, transplantation of CDX2-modulated HE cells represents an effective therapeutic approach for liver injury treatment.