Combinatorial microenvironmental regulation of liver progenitor differentiation by Notch ligands, TGFβ, and extracellular matrix.

The bipotential differentiation of liver progenitor cells underlies liver development and bile duct formation as well as liver regeneration and disease. TGFβ and Notch signaling are known to play important roles in the liver progenitor specification process and tissue morphogenesis. However, the complexity of these signaling pathways and their currently undefined interactions with other microenvironmental factors, including extracellular matrix (ECM), remain barriers to complete mechanistic understanding. Utilizing a series of strategies, including co-cultures and cellular microarrays, we identified distinct contributions of different Notch ligands and ECM proteins in the fate decisions of bipotential mouse embryonic liver (BMEL) progenitor cells. In particular, we demonstrated a cooperative influence of Jagged-1 and TGFβ1 on cholangiocytic differentiation. We established ECM-specific effects using cellular microarrays consisting of 32 distinct combinations of collagen I, collagen III, collagen IV, fibronectin, and laminin. In addition, we demonstrated that exogenous Jagged-1, Delta-like 1, and Delta-like 4 within the cellular microarray format was sufficient for enhancing cholangiocytic differentiation. Further, by combining Notch ligand microarrays with shRNA-based knockdown of Notch ligands, we systematically examined the effects of both cell-extrinsic and cell-intrinsic ligand. Our results highlight the importance of divergent Notch ligand function and combinatorial microenvironmental regulation in liver progenitor fate specification.