背景:人诱导多能干细胞(hiPSCs)及其分化的细胞类型具有很大的组织修复和再生潜力。虽然历史上使用hiPSC的主要焦点是再生受损组织,新出现的研究表明,hiPSC衍生的旁分泌因子对组织再生有更有效的作用.然而,移植的hiPSC来源的细胞分泌组的精确含量不明确。这主要是由于缺乏在体内复杂的组织微环境中区分细胞特异性分泌组和宿主来源的蛋白质的工具。
方法:在本研究中,我们提出了一个新的hiPSC系的产生和表征,L274G-hiPSC,表达鼠突变型甲硫氨酰-tRNA合成酶,L274GMmMetRS,可用于通过双正交非规范氨基酸标记(BONCAT)跟踪细胞特异性蛋白质组。我们在体外和体内评估了L274G-hiPSC的三系分化潜力。此外,我们评估了L274G-hiPSC来源的心肌细胞(L274G-hiPSC-CMs)在体外与野生型人脐静脉来源的内皮细胞共培养后以及在小鼠心脏移植后体内的细胞特异性蛋白质组标记.
结果:我们证明了L274G-hiPSC表现出典型的hiPSC特征,并且我们可以有效地追踪属于三个胚系的分化后代中的细胞特异性蛋白质组,包括L274G-hiPSC-CM。最后,我们在移植的L274G-hiPSC-CM中证明了细胞特异性BONCAT。
结论:新型L274G-hiPSC细胞系可用于体外和体内研究hiPSC的细胞特异性蛋白质组,描述基于hiPSC的细胞疗法用于各种再生医学应用的潜在机制。
BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo.
METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts.
RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs.
CONCLUSIONS: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.