Background: Fully understanding the developmental process of hepatic stem cells (HSCs) and the mechanisms of their committed differentiation is essential for optimizing the generation of functional hepatocytes for cell therapy in liver disease. Delta-like 1 homolog (Dlk1), primarily the membrane-bound form (Dlk1M), is generally used as a surface marker for fetal hepatic stem cell isolation, while its soluble form (Dlk1S) and the functional roles of different Dlk1 isoforms in HSC differentiation remain to be investigated.
Methods: Hepatic spheroid-derived cells (HSDCs) were isolated from E12.5 mouse livers to obtain Dlk1+ and Dlk1-subpopulations. Colony formation, BrdU staining, and CCK8 assays were used to evaluate the cell proliferation capacity, and hepatic/cholangiocytic differentiation and osteogenesis/adipogenesis were used to assess the multipotency of the two subpopulations. Transformation of Dlk1+ cells into Dlk1- cells was detected by FACS, and the expression of Dlk1 isoforms were measured by western blot. The distinct roles and regulatory mechanisms of Dlk1 isoforms in HSC differentiation were investigated by overexpressing Dlk1M.
Results: HSDCs were capable of differentiating into liver and mesenchymal lineages, comprising Dlk1+ and Dlk1- subpopulations. Dlk1+ cells expressed both Dlk1M and Dlk1S and lost expression of Dlk1M during passaging, thus transforming into Dlk1- cells, which still contained Dlk1S. Dlk1- cells maintained a self-renewal ability similar to that of Dlk1+ cells, but their capacity to differentiate into cholangiocytes was obviously enhanced. Forced expression of Dlk1M in Dlk1- cells restored their ability to differentiate into hepatocytes, with an attenuated ability to differentiate into cholangiocytes, suggesting a functional role of Dlk1 in regulating HSC differentiation in addition to acting as a biomarker. Further experiments illustrated that the regulation of committed HSC differentiation by Dlk1 was mediated by the AKT and MAPK signaling pathways. In addition, bFGF was found to serve as an important inducement for the loss of Dlk1M from Dlk1+ cells, and autophagy might be involved.
Conclusions: Overall, our study uncovered the differential expression and regulatory roles of Dlk1 isoforms in the commitment of HSC differentiation and suggested that Dlk1 functions as a key regulator that instructs cell differentiation rather than only as a marker of HSCs. Thus, our findings expand the current understanding of the differential regulation of bi-potential HSC differentiation and provide a fine-tuning target for cell therapy in liver disease.
Keywords: Differentiation; Dlk1; Hepatic stem cells; Isoforms.