Directing stem cell fate using tuneable biomimetic peptide hydrogels

组织硬度在控制细胞功能，特别是指导干细胞命运方面起着至关重要的作用，开创性研究表明，通过将基质硬度与组织硬度相匹配，成人间充质干细胞（MSC）分化可以沿着离散谱系定向，例如，在坚硬基质上的骨生成与在柔软基质上的神经生成。这种材料诱导的谱系承诺为理解机械传感/机械转导机制以及基于细胞的组织工程和再生医学疗法提供了巨大的潜力。然而，常用的2D基板不提供3D体内微环境的真实表示。因此，该项目将采用基于自组装肽的机械可调生物材料，这提供了一个更加仿生的环境，以研究刚度对MSC命运的影响。水凝胶将与Biogelx合作设计，合成和表征，Biogelx是一家专门开发化学和机械可调自组装肽水凝胶的公司，具有与人体组织相似的纳米级结构，其在细胞培养/生物学和细胞-基于再生疗法。该项目将重点阐明如何利用水凝胶硬度来引导MSC谱系向柔软和光滑的方向发展。硬组织表型所产生的数据将增强对组织硬度对MSC命运影响的生物学原理的理解，并使未来使用水凝胶的再生疗法的发展成为可能。（原子力显微镜、流变学、SEM和质谱）; MSC表型分析（定量PCR、多参数流式细胞术、蛋白质印迹、免疫荧光）;转录组学（RNA-Seq）;细胞转化和实时共聚焦活细胞成像。Richardson SM，Hughes N，Hunt JA，Freemont AJ，Hoyland JA.壳聚糖-甘油磷酸盐水凝胶中人间充质干细胞向NP样细胞的分化。生物材料。2008;29（1）：85-93. Jayawarna V，Richardson SM，Hirst AR，Hodson NW，Saiani A，Gough JE，Ulijn RV.在用于细胞培养的Fmoc-肽凝胶中引入化学功能。生物材料学报. 2009;5（3）：934- 43. Engler AJ，Sen S，Sweeney HL，Discher D.E.基质弹性指导干细胞谱系特化。Cell. 2006;126（4）：677- 89. Swift，J.，伊万诺夫斯卡岛Buxboim，A.，Harada，T.，Dingal，P.，Pinter，J.，Pajerowski，J.，Spinler，K.，申，J. Tewari，M. & Discher，D.E.核层蛋白-A与组织硬度成比例，并增强基质定向分化。科学2013;341（6149）：1240104

Tissue stiffness plays a crucial role in controlling cell function and, in particular, directing stem cell fate, with seminal studies demonstrating that by matching substrate stiffness to tissue stiffness, adult human mesenchymal stem cell (MSC) differentiation can be directed down discrete lineages e.g. osteogenesis on stiff substrates vs. neurogenesis on soft substrates. This material-induced lineage commitment offers huge potential both for understanding mechanosensing/mechanotransduction mechanisms and for cell-based tissue engineering and regenerative medicine therapies. However, commonly used 2D substrates do not offer a true representation of the 3D in vivo microenvironment. Therefore this project will employ self-assembling peptide-based mechanically-tuneable biomaterials, which offer a more biomimetic environment in which to study the impact of stiffness on MSC fate. Hydrogels will be designed, synthesised and characterised in collaboration with Biogelx, a company specialising in development of chemically and mechanically tuneable self-assembling peptide hydrogels with similar nanoscale structures to human tissues, which have a wide range of applications in cell culture/biology and cell-based regenerative therapies.The project will focus on elucidating how hydrogel stiffness can be used to direct MSC lineage commitment towards soft and hard tissue phenotypes. The data generated will enhance understanding of the biological principles underpinning tissue stiffness effects on MSC fate and enable development of future regenerative therapies using hydrogels.The project will offer state-of-the-art multidisciplinary training, including biomaterial characterisation methods (atomic force microscopy, rheology, SEM and mass spectrometry); MSC phenotyping (quantitative PCR, multi-parametric flow cytometry, western blotting, immunofluorescence); transcriptomics (RNA-Seq); cell transformation and real-time confocal live-cell imaging.Richardson SM, Hughes N, Hunt JA, Freemont AJ, Hoyland JA. Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels. Biomaterials. 2008;29(1):85-93. Jayawarna V, Richardson SM, Hirst AR, Hodson NW, Saiani A, Gough JE, Ulijn RV. Introducing chemical functionality in Fmoc-peptide gels for cell culture. Acta Biomaterialia. 2009;5(3):934-43.Engler AJ, Sen S, Sweeney HL, Discher D.E. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-89.Swift, J., Ivanovska, I., Buxboim, A., Harada, T., Dingal, P., Pinter, J., Pajerowski, J., Spinler, K., Shin, J., Tewari, M. & Discher, D.E. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science. 2013;341(6149):1240104