The impact of mesenchymal stromal cells on the proliferation and functional maturation of liver organoids

The health burden of end-stage liver disease continues to grow, and orthotopic liver transplantation remains the only curative treatment. Three-dimensional (3D) culture systems support the in vitro culture of primary liver cells, allowing them to recapitulate native tissue architecture. This technology holds considerable potential for regenerative medicine and disease modeling. However, the practical application of liver organoid (LO) technology is hindered by the inherent limitations of LOs derived from single-tissue sources. In this study, we integrated human umbilical cord mesenchymal stromal cells (hUC-MSCs) into single-source LOs and assessed their effects on organoid formation and function. During the expansion phase, co-culture with hUC-MSCs yielded a 2-fold increase in organoid number compared to LOs cultured alone, although no significant changes were observed in the expression of genes related to organoid lifespan. Liver-specific functions of LOs after 7 day incubated with differentiation medium were further investigated. Despite constructing organoids from mature hepatocytes, co-culture with MSCs promoted non-hepatocyte differentiation, as evidenced by the expression of the cholangiocyte organoid marker cytokeratin 19. Furthermore, LOs co-culture with hUC-MSCs demonstrated increased albumin and urea secretion. In contrast, glucose consumption and ammonia clearance rates showed no significant differences. Following in vivo transplantation, serum biochemical markers normalized in both transplanted and control groups. However, histological analysis revealed superior liver tissue repair in the transplanted group. Our findings indicate that MSCs positively enhance the proliferative capacity of LOs and confer limited functional benefits, and incorporation of MSCs may promote non-hepatic cell differentiation. This effect was particularly evident within the extracellular matrix components of the hydrogel culture system.