Well-defined, modifiable hydrogel networks to unravel key parameters that control stem cell fate in the bone marrow.

骨髓中存在两种成体干细胞：骨髓基质/间充质干细胞（MSCs）和造血干细胞。骨髓有一个独特的机械环境，它被生理活动和废弃，以及对疾病和衰老的反应所调节。人体内的许多细胞，包括干细胞，都具有高度的机械反应性。已知的直接干细胞命运的物理特性包括地形、硬度、粘附配体的间距和聚集。例如，在不同硬度的表面上培养MSCs，在缺乏可溶性因子的情况下，它们的命运将沿着神经源性、肌源性和成骨性谱系发展。然而，这些现象只在二维表面上培养的细胞中进行了系统的探索，这并不能复制天然组织的三维环境。有许多关于骨髓的整体力学性能的报道，这些力学性能是通过测量静水压力和粘度来确定的。然而，我们对骨髓的局部力学特性知之甚少，在这个尺度上，细胞可以机械地检测其局部环境的刚度。此外，考虑到骨髓这些3D物理特性的体外模型使研究人员能够提出一些基本问题，如硬度等因素在指导干细胞命运和维持健康和疾病中的造血方面的影响。为了解决这些问题，我们将使用基于荧光显微镜和原子力显微镜（AFM）的微压痕的组合来表征小鼠骨髓中干细胞生态位的机械环境。绅士实验室开发了一种新型的聚乙二醇肽水凝胶，其中水凝胶化学的系统修饰允许精确控制水凝胶的物理性质，如刚度，粘附配体定位和凝胶可降解性。聚乙二醇肽水凝胶也具有生物相容性，允许活细胞的包封，使这些凝胶成为体外模拟骨髓的理想系统。因此，我们还将制造具有与天然骨髓相匹配的刚度的peg肽水凝胶，包封活细胞，并研究骨髓物理和机械特性的变化如何影响天然组织样壁龛中的干细胞反应。这个跨学科项目融合了KCL， UCL和Imperial在聚合物合成，机械生物学，肽化学，干细胞生物学和力学方面的专业知识，以确定骨髓干细胞生态位的机械特性，然后基于定义良好的肽修饰水凝胶创建一个体外模型，该水凝胶具有可修改的物理和生物特性，可以模仿它。有了这个模型，我们将提出关于干细胞如何对其环境的物理特性作出反应的基本问题。总的来说，这个项目应该为干细胞力学生物学和骨髓干细胞生态位在健康和疾病中的物理特性的作用提供基本的见解。

Two adult stem cell populations reside in the bone marrow: marrow stromal/mesenchymal stem cells (MSCs) and haematopoietic stem cells. The marrow has a unique mechanical environment, which is known to be modulated by physiological activity and disuse, as well as in response to disease and aging. Many cells in the body, including stem cells, are highly mechanoresponsive. Physical characteristic that are known to direct stem cell fate include topography, stiffness and the spacing and clustering of adhesive ligands. For example, culture of MSCs on surfaces of different stiffnesses will direct their fate down neurogenic, myogenic and osteogenic lineages in the absence of soluble factors. However, these phenomena have only been systemically explored in cells cultured on 2D surfaces, which do not replicate the 3D environment of native tissues. There are a number of reports on the bulk mechanical properties of bone marrow that have been determined from measurements of hydrostatic pressure and viscosity. However, very little is known about the local mechanical properties of the marrow at the scale at which a cell mechanically detects the stiffness of its local environment. Moreover, in vitro models that account for these 3D physical properties of the marrow allow researchers to ask fundamental questions on the effect of factors such as stiffness in directing stem cell fate and in the maintenance of haematopoiesis in health and disease. To address these questions, we will use a combination of fluorescent microscopy and atomic force microscopy (AFM)-based microindentation to characterise the mechanical environment of the stem cell niche in mouse bone marrow. The Gentleman lab has developed a novel PEG-peptide hydrogel, in which systematic modifications of hydrogel chemistry allow for precise control of hydrogel physical properties such as stiffness, adhesive ligand positioning and gel degradability. PEG-peptide hydrogels are also biocompatible, allowing for encapsulation of live cells, making these gels an ideal system to mimic the bone marrow in vitro. Therefore, we will also create PEG-peptide hydrogels with stiffnesses that match those of native bone marrow, encapsulate live cells, and examine how changes in the physical and mechanical properties on the marrow affects stem cell response in native tissue-like niches. This interdisciplinary project melds expertise in polymer synthesis, mechanobiology, peptide chemistry, stem cell biology and mechanics at KCL, UCL and Imperial to determine the mechanical properties of the bone marrow stem cell niche, and then create an in vitro model based on well-defined, peptide-modified hydrogels with modifiable physical and biological properties that mimics it. With this model, we will ask fundamental questions regarding how stem cells respond to physical properties of their environment. Overall, this project should provide fundamental insights into stem cell mechanobiology and the role of physical properties in the bone marrow stem cell niche in health and disease.