Distinct roles of Dlk1 isoforms in bi-potential differentiation of hepatic stem cells
Distinct roles of Dlk1 isoforms in bi-potential differentiation of hepatic stem cells
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Dlk1亚型在肝干细胞双能分化中的独特作用
DOI：
10.1186/s13287-019-1131-2
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发表时间：
2019-01-15
影响因子：
7.5
通讯作者：
Xu, Huanbai
中科院分区：
文献类型：
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作者：
Huang, Jiefang;Zhao, Xiaonan;Xu, Huanbai
全面了解肝干细胞(HSCs)的发育过程及其定向分化机制对于优化肝病细胞治疗所需的功能性肝细胞的生成至关重要。Delta样1同系物(Dlk1)，主要是膜结合型(Dlk1M)，通常被用作胎肝干细胞分离的表面标志，但其可溶性形式(Dlk1S)及其不同亚型在肝星状细胞分化中的作用仍有待研究。方法从E12.5小鼠肝脏分离肝球形衍生细胞(HSDCs)，获得Dlk1+和Dlk1−亚群。集落形成、BrdU染色和CCK8检测细胞增殖能力，肝/胆管细胞分化和成骨/成脂能力检测两个亚群的多能性。流式细胞仪检测Dlk1+细胞向Dlk1−细胞的转化，免疫印迹法检测Dlk1亚型的表达。结果HSDCs可分化为肝细胞和间充质细胞，包括Dlk1+和Dlk1−亚群。Dlk1+细胞同时表达Dlk1M和Dlk1S，并在传代过程中失去Dlk1M的表达，从而转化为Dlk1−细胞，但仍含有Dlk1S。DLK1−细胞保持与DLK1+细胞相似的自我更新能力，但其向胆管细胞分化的能力明显增强。Dlk1Min Dlk1HSC的强制表达恢复了其向肝细胞分化的能力，但向胆管细胞分化的能力减弱，提示Dlk1除了作为生物标志物外，还在调节−分化方面发挥作用。进一步的实验表明，Dlk1对承诺的HSC分化的调控是通过AKT和MAPK信号通路介导的。结论本研究揭示了Dlk1亚型在HSC分化过程中的差异表达和调控作用，提示Dlk1不仅是HSC分化的标志物，而且是指导细胞分化的关键调节因子。因此，我们的发现扩大了目前对双潜能HSC分化差异调控的理解，并为肝病的细胞治疗提供了一个微调的靶点。
BackgroundFully 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.MethodsHepatic 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.ResultsHSDCs were capable of differentiating into liver and mesenchymal lineages, comprising Dlk1+and Dlk1−subpopulations. Dlk1+cells expressed both Dlk1Mand Dlk1Sand lost expression of Dlk1Mduring 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 Dlk1Min 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 Dlk1Mfrom Dlk1+cells, and autophagy might be involved.ConclusionsOverall, 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.