Modulation of limbal niche stiffness to regulate stem cell differentiation

组织工程学是一种新兴的生物医学方法，主要基于干细胞的自然修复能力来帮助和加速缺陷和受损组织的再生。对于这一新的治疗策略，为细胞提供促进和调节其增殖和分化的局部环境(利基)是必不可少的。在这方面，生物材料技术目前处于领先地位，试图通过重建人工三维环境来满足这些要求。然而，了解这些细胞是如何、为什么以及在什么环境中以特定于谱系的方式分化的，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(即使细胞做出反应的特定信号或刺激)尚未得到适当的研究，这可能会破坏目前利用生物材料重建干细胞生态位的尝试。因此，我们计划了解基质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基质硬度相关(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眼表生物材料和角膜干细胞移植的MRC、BBSRC和EPSRC研究。声致非弹性光散射(布里渊显微镜)可以非接触、直接读出材料的机械性能，并已成功地用于在微观分辨率下测量人眼角膜和晶状体的生物力学性能。我们计划使用这种复杂的方法来测量正常和疾病/受伤的角膜表面的硬度，生成组织机械特性的高分辨率地图。然后，我们将寻求使用一种酶(胶原酶)以高度可控的方式分解角膜主要结构蛋白(胶原)来影响局部僵硬。这种酶将通过一种粘在角膜上的凝胶传递，以实现酶的精确定位和相应的局部僵硬效果。我们之前已经证明，角膜干细胞在硬质胶原胶上生长时会分化，胶原酶可以通过分化细胞的比例减少来减少这种僵硬。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硬度变化敏感之后，我们将开发一种方法，在角膜干细胞移植之前控制其硬度，相信这将导致移植细胞以未分化形式延长驻留时间。预测的结果是角膜干细胞移植的成功率显著提高。近些年来，角膜干细胞移植的总体临床成功率一直保持在约50%的水平。75%的人能够辨别出眼表上两行或两行以上的符号。虽然这是一项了不起的外科成就，但仍有相当大的改进空间。我们认为，在干细胞移植前恢复角膜组织的机械性能是一种显著的外科增强。

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.