背景:已证明干细胞微环境强烈影响其生物学功能和临床分级。天然或合成生长因子,尤其是,对调节干细胞增殖至关重要,新陈代谢,并通过与特定的细胞外受体相互作用而分化。成纤维细胞生长因子-2(FGF-2)在各种组织和器官中具有多效性功能。它与FGF受体(FGFR)相互作用并激活FGFR信号通路,涉及许多生物学功能,比如血管生成,伤口愈合,细胞增殖,和差异化。
目标:这里,我们的目标是探索分子功能,行动模式,和功能尚未确定的治疗活性,FGF-2衍生肽,FP2(44-ERGVVSIKGV-53)促进增殖,分化,和治疗应用的沃顿的果冻间充质干细胞(WJ-MSCs)相比,其他测试肽,canof1(FP1),hexafin2(FP3),和canofin3(FP4)具有已知功能。
方法:通过EDC/NHS化学将与贻贝粘附蛋白(MAP)融合的测试肽固定在培养板上。细胞增殖试验,菌落形成单位,蛋白质印迹分析,基因表达分析,RNA-Seq.分析,成骨,和软骨分化能力用于测试测试肽的活性。我们还利用三维(3D)结构分析和基于人工智能(AI)的AlphaFold2和CABS-dock程序来预测肽受体的受体相互作用。我们还使用骨关节炎小鼠模型验证了FP2培养的WJ-MSC的体内治疗能力。
结果:与测试肽相比,将WJ-MSC培养到FP2固定的培养板上显示细胞增殖(n=3;*p<0.05,**p<0.01)和集落形成单位(n=3;*p<0.05,**p<0.01)的显著增加。FP2显示FRS2α和FGFR1的磷酸化显著上调,并激活AKT和ERK信号通路(n=3;*p<0.05,**p<0.01,***p<0.001)。有趣的是,我们检测到使用基于AI的工具预测的有效FP2受体结合。用AKT抑制剂处理显著消除了FP2介导的细胞分化增强(n=3;*p<0.05,**p<0.01,***p<0.001)。在骨关节炎小鼠模型中,关节内注射FP2培养的MSCs可显着减轻关节炎症状,如功能测试所示(n=10;*p<0.05,**p<0.01,***p<0.001,***p<0.0001),调节促炎和抗炎基因的表达水平,和改善骨软骨再生,如组织切片所示。
结论:我们的研究确定FGF-2衍生肽FP2是一种有希望的候选肽,可以提高WJ-MSCs的治疗潜力,尤其是骨和软骨的再生。
BACKGROUND: The stem cell microenvironment has been evidenced to robustly affect its biological functions and clinical grade. Natural or synthetic growth factors, especially, are essential for modulating stem cell proliferation, metabolism, and differentiation via the interaction with specific extracellular receptors. Fibroblast growth factor-2 (FGF-2) possesses pleiotropic functions in various tissues and organs. It interacts with the FGF receptor (FGFR) and activates FGFR signaling pathways, which involve numerous biological functions, such as angiogenesis, wound healing, cell proliferation, and differentiation.
OBJECTIVE: Here, we aim to explore the molecular functions, mode of action, and therapeutic activity of yet undetermined function, FGF-2-derived peptide, FP2 (44-ERGVVSIKGV-53) in promoting the proliferation, differentiation, and therapeutic application of human Wharton\'s jelly mesenchymal stem cells (hWJ-MSCs) in comparison to other test peptides, canofin1 (FP1), hexafin2 (FP3), and canofin3 (FP4) with known functions.
METHODS: The immobilization of test peptides that are fused with mussel adhesive proteins (MAP) on the culture plate was carried out via EDC/NHS chemistry. Cell Proliferation assay, colony-forming unit, western blotting analysis, gene expression analysis, RNA-Seq. analysis, osteogenic, and chondrogenic differentiation capacity were applied to test the activity of the test peptides. We additionally utilized three-dimensional (3D) structural analysis and artificial intelligence (AI)-based AlphaFold2 and CABS-dock programs for receptor interaction prediction of the peptide receptor. We also verified the in vivo therapeutic capacity of FP2-cultured hWJ-MSCs using an osteoarthritis mice model.
RESULTS: Culture of hWJ-MSC onto an FP2-immobilized culture plate showed a significant increase in cell proliferation (n = 3; *p < 0.05, **p < 0.01) and the colony-forming unit (n = 3; *p < 0.05, **p < 0.01) compared with the test peptides. FP2 showed a significantly upregulated phosphorylation of FRS2α and FGFR1 and activated the AKT and ERK signaling pathways (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Interestingly, we detected efficient FP2 receptor binding that was predicted using AI-based tools. Treatment with an AKT inhibitor significantly abrogated the FP2-mediated enhancement of cell differentiation (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Intra-articular injection of FP2-cultured MSCs significantly mitigated arthritis symptoms in an osteoarthritis mouse model, as shown through the functional tests (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), modulation of the expression level of the pro-inflammatory and anti-inflammatory genes, and improved osteochondral regeneration as demonstrated by tissue sections.
CONCLUSIONS: Our study identified the FGF-2-derived peptide FP2 as a promising candidate peptide to improve the therapeutic potential of hWJ-MSCs, especially in bone and cartilage regeneration.