A 2D‑3D culture transition enhances hepatic maturation of human mesenchymal stem cell-derived hepatocyte-like cells

Hepatocyte-like cells (HLCs) derived from mesenchymal stem cells (MSCs) hold considerable promise for drug screening and regenerative therapy, but their clinical translation is limited by functional immaturity. This study aimed to design and evaluate a novel hepatic differentiation strategy to address this limitation and enhance the application of HLCs in preclinical drug development and cell therapy for liver disease. We developed a dimensional transition strategy that shifts human MSCs (hMSCs) from 2D monolayer differentiation to 3D spheroid culture (termed 2D-3D transition) to promote hMSCs hepatic differentiation. The phenotypes and functions of differentiated HLCs were characterized to assess the efficiency of hMSCs hepatic differentiation. Dimensionally transitioned HLCs encapsulated in Pluronic F-127 hydrogel were transplanted into acute liver failure (ALF) mice to evaluate the therapeutic efficacy. The mechanism of promoting hMSCs hepatic differentiation focused on the mechanosensitive receptor Piezo1 and its downstream epigenetic regulator signals. This approach significantly enhanced hepatic maturation-associated phenotype of HLCs, as demonstrated by reduced expression and secretion of alpha fetoprotein (AFP)–a hallmark of fetal hepatocytes. Transcriptional profile results revealed transcriptomic similarity between 2D-3D HLCs and primary human hepatocytes (PHHs). Furthermore, 2D-3D HLCs exhibited drug sensitivity profiles comparable to hepatocytes and showed markedly upregulated CYP3A4 mRNA expression and enzyme activity. In the ALF model, transplantation of 2D-3D HLCs significantly improved survival rates and alleviated liver injury. Mechanistically, the 2D-3D transition was associated with the involvement of Piezo1-associated p300/H3K56ac signaling. This study not only presents a novel strategy for generating HLCs with enhanced hepatic maturation-associated phenotypes, but also provides the potential for efficient generation of HLCs and a better understanding of hMSCs hepatic differentiation for future therapeutic explorations.