Modulation of limbal niche stiffness to regulate stem cell differentiation

组织工程是一种新兴的生物医学方法，主要基于干细胞的自然愈合潜力来帮助和加速缺陷和受损组织的再生。对于这种新的治疗策略，为细胞提供一个增强和调节其增殖和分化的局部环境（生态位）是必不可少的。在这方面，生物材料技术目前在试图通过重建人工三维环境来满足这些要求方面处于领先地位。然而，了解这些细胞如何、为什么以及在什么环境下以谱系特异性方式分化对于组织工程和干细胞治疗至关重要。我们认为，这种潜在的生物学（即使细胞作出反应的特定信号或刺激）尚未得到适当的研究，这可能会破坏目前使用生物材料重建干细胞生态位的尝试。因此，我们计划了解底物硬度在维持角膜干细胞生态位（覆盖角膜表面所需细胞的家园）中所起的作用。我们在眼表模型方面的专业知识意味着我们能够很好地研究与底物刚度依赖（机械敏感）分化相关的基因和蛋白质表达的变化。这些研究将直接进入组织工程领域，以及我们自己正在进行的MRC、BBSRC和EPSRC对眼表生物材料和角膜干细胞移植的研究。声诱导非弹性光散射（布里渊显微镜）允许非接触，直接读出材料的机械性能，并已成功地用于测量，在微观分辨率下，人眼角膜和晶状体的生物力学特性。我们计划采用这种复杂的方法来测量正常和疾病/受伤角膜表面的刚度，从而产生组织机械特性的高分辨率图。然后，我们将寻求用一种酶（胶原酶）以高度控制的方式分解角膜的主要结构蛋白（胶原蛋白）来影响局部僵硬。这种酶将通过粘在角膜上的凝胶来输送，从而使酶能够精确定位，并对硬度产生相应的局部影响。我们之前已经证明，角膜干细胞在坚硬的胶原凝胶上生长时会分化，胶原酶可以通过比例减少分化细胞来降低这种硬度。因此，我们计划研究一种新的角膜前干细胞移植技术。简而言之，在验证了角膜干细胞生态位对其刚度变化的敏感性之后，我们将开发一种在角膜干细胞移植之前控制其刚度的方法，并相信这将导致移植细胞在未分化形式下的驻留时间的改善。预测的结果是角膜干细胞移植的成功率显著提高。近年来，角膜干细胞移植的整体临床成功率一直保持稳定在约。75%的人能够分辨出视力表上的两条线或更多的符号。虽然这是一项显著的手术成就，但仍有很大的改进空间。我们认为，在干细胞移植之前恢复角膜组织的力学特性是一种显著的手术增强。

Tissue engineering is a newly emerging biomedical methodology to assist and accelerate the regeneration of defective and damaged tissues based largely on the natural healing potentials of stem cells. For this new therapeutic strategy, it is indispensable to provide cells with a local environment (niche) that enhances and regulates their proliferation and differentiation. In this regard, biomaterial technology currently leads the way in trying to fulfil these requirements by recreating artificial three-dimensional environments. However, understanding how, why and in what environment these cells differentiate in a lineage-specific manner are essential for tissue engineering and stem cell therapy. We believe that this underlying biology (i.e. specific signals or stimuli which make the cells respond) has not yet been properly investigated and that this is likely to undermine current attempts to recreate the stem cell niche using biomaterials. Therefore, we plan to understand the role substrate stiffness plays in maintaining the corneal stem cell niche (the home for cells required to cover the surface of the cornea). Our expertise in ocular surface models means we are well placed to investigate the changes in gene and protein expression associated with substrate stiffness dependent (mechanosensitive) differentiation. Together these investigations will feed directly into the field of tissue engineering and our own on-going MRC, BBSRC and EPSRC research into ocular surface biomaterials and corneal stem cell transplantation. Acoustically induced inelastic light scattering (Brillouin microscopy) allows non-contact, direct readout of the mechanical properties of a material and has been used successfully to measure, at microscopic resolution, the biomechanical properties of the cornea and lens of the human eye. We plan to employ this sophisticated method to measure stiffness across the surface of normal and disease/wounded corneas producing high resolution maps of the tissues' mechanical properties. We will then seek to affect localised stiffness using an enzyme (collagenase) to break up the corneas main structural protein (collagen) in a highly controlled manner. The enzyme will be delivered by a gel that sticks to the cornea to allow precise positioning of the enzyme and a corresponding localised effect on stiffness. We have shown previously that corneal stem cells differentiate when grown on stiff collagen gels and that collagenase can reduce this stiffness with a proportional reduction in differentiated cells. Therefore we plan to investigate a new pre-corneal stem cell transplantation technique. In short, having validated that the corneal stem cell niche is sensitive to changes in its stiffness we will develop a method to control its stiffness prior to corneal stem cell transplantation with the belief that this will result in an improved residency time for the transplanted cells in an undifferentiated form. The predicted outcome being a significantly improved success rate for corneal stem cell transplantation. Recently the overall clinical success rate of corneal stem cell transplantation has remained static at approx. 75% with half of those able to discern two lines or more of symbols on an eye chart. Whilst this represents a remarkable surgical achievement there remains considerable room for improvement. We believe restoring the corneal tissues mechanical properties prior to stem cell transplantation represents a significant surgical enhancement.