OPTIMISING STEM CELL THERAPY: INVESTIGATING CLONAL HETEROGENEITY IN EQUINE CHONDROPROGENITOR CELLS

在马和其他脊椎动物的关节中，骨骼覆盖着软骨，软骨提供了一个光滑、低阻力、承重的表面，使骨骼能够以最小的阻力相互移动。在受伤时，软骨的自我修复能力有限，因为它没有血液或神经供应。马的腕关节损伤（或“缺陷”）可以通过关节的疾病和创伤性损伤发生。如果不及时治疗，这些缺陷可能导致骨关节炎。在马和人类中，软骨细胞疗法是用于替换受伤或骨关节炎软骨的技术，但它是不可靠的。我们知道修复不可靠的一些原因，其中一个主要原因与所使用的细胞有关。通常，用于该过程的细胞取自接受治疗的患者的软骨。不幸的是，当这些细胞（“软骨细胞”）在关节外停留太长时间时，它们就失去了变回软骨细胞的能力，并停止增殖。在我们的实验室中，在马软骨中发现了一种祖细胞。祖细胞类似于干细胞，并且具有成为许多不同细胞/组织类型的能力。我们已经证明，软骨祖细胞不会遭受与软骨细胞相同的问题。它已经表明，你可以在实验室中产生数亿个祖细胞（以快速的速度），它们将在需要时可靠地转化为软骨细胞。这些属性使祖细胞成为修复马关节软骨的上级候选细胞。干细胞和祖细胞一旦从其宿主组织（例如骨髓）中分离出来，就会形成集落。在实验室的塑料培养皿中将大量菌落一起生长，直到达到足够数量的细胞以提供治疗。然而，人们普遍认为，干细胞治疗的结果是巨大的变化，这被认为是部分原因，由于缺乏均匀性的干细胞和他们形成的集落。最近的工作在我们的实验室已经证明，祖细胞集落从马软骨也缺乏均匀性。不仅菌落看起来不同，而且还观察到，当它们作为单个单独的菌落生长时，它们繁殖的速率以及它们形成的组织工程软骨的质量和数量都存在差异。目前，我们还没有一种精确的方法来选择“正确”的集落，以提供细胞进行治疗。我们也不知道是否可以将多个集落一起生长（类似于从骨髓中生长干细胞），或者集落是否应该作为单个单独的集落生长。许多菌落一起生长可能会引起正反馈或负反馈;即“坏”菌落会降低“好”菌落的质量，反之亦然。因此，本研究的目的是：1）研究菌落形状和结构的重要性。我们是否能够根据它们形成的集落类型选择好的细胞进行治疗？2)比较和对比单个菌落和多个菌落（一起生长的菌落）的软骨质量。我们还将确定将不同已知质量的单菌落混合在一起的效果。3）研究来自单菌落的细胞的物理性质。我们是否能够根据细胞的物理特性选择好的细胞进行治疗？这项研究的数据将使我们能够确定为马的软骨修复提供最大潜力的最佳菌落。了解干细胞和祖细胞集落缺乏均匀性具有重要的临床意义，因为这些数据还将帮助我们确定，出于治疗目的，单克隆或多克隆方法是否是改善马治疗临床结果的长期目标的最佳方法。

In the joints of horses other vertebrates, bones are covered with cartilage which provides a smooth, low resistance, weight bearing surface that enables the bones to move against each other with minimal resistance. Upon injury, cartilage has a limited capacity for self repair as it does not have a blood or nerve supply. Cartilage damage (or 'defects') in the horse can occur through both disease and traumatic injury to the joint. If left untreated, the defects can lead to osteoarthritis. In horses and humans, cartilage cell therapy is a technique utilised to replace injured or osteoarthritic cartilage but it is unreliable. We know some of the reasons why repair is unreliable, and a major one relates to the cells used. Generally the cells used for this procedure are taken from cartilage of the patient undergoing treatment. Unfortunately, when these cells ('chondrocytes') spend too much time out of the joint they lose their ability to turn back into cartilage cells and stop multiplying.In our laboratory, a progenitor cell has been discovered in horse cartilage. Progenitor cells are similar to stem cells and have the capacity to become a number of different cell/tissue types. We have demonstrated that cartilage progenitor cells don't suffer from the same problems as chondrocytes. It has been shown that you can generate hundreds of millions of progenitor cells in the laboratory (at a fast rate) and they will reliably turn into cartilage cells when required. These attributes make progenitor cells superior candidates for repairing the cartilage in horse joints. It is the nature of stem and progenitor cells, once isolated from their host tissue (for example, bone marrow), to form colonies. Numerous colonies are grown together in plastic dishes in the laboratory until a sufficient number of cells is reached to provide treatment. However, it is widely accepted that the results of stem cell treatment are hugely variable and this is believed to be in part, due to the lack of uniformity in stem cells and the colonies they form.Recent work in our laboratory has demonstrated that progenitor cell colonies from horse cartilage also lack uniformity. Not only can the colonies look different but it is also observed that when they are grown as single, separate colonies, there is a difference in the rate at which they multiply and the quality and quantity of tissue engineered cartilage they form. At this present time we do not have a precise method to choose the 'correct' colony in order to provide cells for treatment. We also do not know whether it is acceptable to grow numerous colonies together (akin to growing stem cells from bone marrow) or whether the colonies should be grown as single, separate colonies. It is possible that growing numerous colonies together may induce either positive or negative feedback; i.e do 'bad' colonies reduce the quality of 'good' colonies and vice versa. This study will therefore aim to:1)Investigate the importance of colony shape and structure. Are we able to pick good cells for treatment based on the type of colony they form?2) Compare and contrast the quality of cartilage from single colonies and from muitiple colonies (colonies grown together). We will also determine the effects of mixing together single colonies of varying known quality.3) Investigate the physical properties of the cells derived from single colonies. Are we able to pick good cells for treatment based on the physical properties of the cells?Data from this study will allow us to determine the best colonies that provide the greatest potential for cartilage repair in the horse. Understanding the lack of uniformity in stem and progenitor cell colonies has great clinical importance as these data will also assist us to determine whether, for therapeutic purposes, a single clone or multiple clone method is the best approach with the long term aim of improving the clinical outcome of treatment in the horse.