背景:源自脐带基质的人骨髓间充质干细胞是一种有前途的治疗资源,它们的分化细胞作为组织再生治疗方法受到关注。然而,来自人脐带基质间充质干细胞(hUCM-MSCs)的分化细胞的医学用途存在局限性,例如有效的区分方法。
方法:为了有效地将hUCM-MSCs分化为肝细胞样细胞,我们使用了ROCK抑制剂,法舒地尔,已知诱导内胚层形成,和明胶,为分化的细胞提供细胞外基质。根据明胶和法舒地尔的组合评估早期的分化效率,进行转录分析。此外,为了证明细胞器状态影响分化,我们进行了转录,层析成像,和线粒体功能分析在肝分化的每个阶段。最后,我们根据mRNA和蛋白质的表达评估肝细胞功能,白蛋白的分泌,和CYP3A4在成熟HLC中的活性。
结果:法舒地尔在hUCM-MSCs中诱导内胚层相关基因(GATA4、SOX17和FOXA2),它还在分化细胞内诱导脂滴(LD)。然而,法舒地尔引起的LD的过度诱导抑制了线粒体功能并阻止了向肝细胞的分化。为了防止过多的LD形成,我们使用明胶作为涂层材料。当hUCM-MSCs在高粘度(1%)明胶涂层的培养皿中被法舒地尔诱导为肝细胞时,与用低粘度(0.1%)明胶处理的那些相比,在高粘度明胶涂层的培养皿上,与肝细胞相关的基因(AFP和HNF4A)显示出显着的上调。此外,其他种系细胞命运,如外胚层和中胚层,在这些条件下被压抑。此外,LD丰度也降低了,而线粒体功能增强。另一方面,与分化的早期阶段不同,低粘度明胶更有效地产生成熟的HLC。在这种情况下,LDs在细胞中的积累受到抑制,线粒体被激活了.因此,源自hUCM-MSC的HLC在低粘度明胶中在遗传和功能上更加成熟。
结论:本研究证明了使用法舒地尔和不同粘度的明胶将hUCM-MSCs分化成肝细胞的有效方法。此外,我们认为,高效的肝分化和从hUCM-MSCs分化的肝细胞的功能不仅取决于遗传变化,还取决于细胞器状态的调节。
BACKGROUND: Human mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods.
METHODS: To effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and
gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of
gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs.
RESULTS: Fasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used
gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%)
gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity
gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin.
CONCLUSIONS: This study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.