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

Reference	BB/J009210/1
Principal Investigator / Supervisor	Professor Charles Archer
Co-Investigators / Co-Supervisors	Dr Helen McCarthy
Institution	Cardiff University
Department	School of Biosciences
Funding type	Research
Value (£)	200,121
Status	Completed
Type	Research Grant
Start date	01/04/2012
End date	31/03/2014
Duration	24 months

Abstract

The overall aim of this project is to investigate clonal heterogeneity in equine articular cartilage progenitor cells and, in turn, determine cell lines with optimum chondrogenicity. Currently, there is a knowledge gap in how to identify which cells, following clonal isolation and expansion, will go on to form cartilage capable of functional repair. Data from this study will allow us to determine optimal cell lines that provide the greatest potency in terms of chondrogenesis. Understanding heterogeneity has great clinical importance and these data will assist us to determine whether, for therapeutic purposes, a single clone or polyclonal method is the best approach. If single colonies prove to be the most suitable approach, these data will also enable us to determine which cell line may provide the greatest reparative potential. We aim to reduce such heterogeneity in equine chondroprogenitor cells and optimise their therapeutic use by fully investigating clonal variation and determining optimum chondrogenicity through studying gene expression, immunohistochemistry, biochemical analysis and nano-mechanics. We, therefore, wish to focus on 3 main aims: 1) Investigating the importance of single colony morphology 2) To isolate clonal (single clone) and polyclonal (multiple colonies/clones) cell lines and to compare and contrast chondrogenicity in addition to determining the effects of mixing single colonies of varying chondrogenicity 3) Investigating nano-mechanical properties of expanded single colonies. Thus, by further characterisation of these cells in vitro, we can better understand the relationship of cartilage progenitor cells in development, proliferation and repair, with the long term aim of improving the clinical outcome. This work will also bear relevance to the setting up of immortal cell lines for potential allogeneic procedures in equine cartilage repair.

Summary

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 lackof 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.

Impact Summary

Who will benefit from this research? The horse is the primary beneficiary of this research as joint disease is common cause of morbidity in equine athletes. It has been stated that osteoarthritis accounts for the retirement of 60% of elite athlete horses and current treatments to repair damaged equine articular cartilage provides a less than favourable outcome with no current therapy resulting in complete restoration of the hyaline cartilage to a normal status. It is anticipated that over 50% of horses with cartilage damage would be amenable to therapy using articular cartilage progenitor cells (Prof. Wayne McIlwraith, personal communication). The vast number of horses in the UK and the US suggest a considerable market for treatment. Human: Even though our current focus is on treatment for equine patients, articular cartilage progenitor cells will be of great use in research targeting cartilage repair in humans. The technology arising from this work will be provide an excellent animal model for the treatment of human joint disease. Indeed, for joint repair procedures, the U.S Food and Drug Administration (FDA) now recommends that the horse be the animal model of choice prior to human clinical trials. Cardiff University: this piece of translational-style funding will greatly benefit the University because as it will create an increase the opportunity for its research to move from lab bench to clinic. It will also allow the Archer lab and the commercialisation division (RACDV) to disseminate information as it is being created to other projects within the University. Colorado State University/Swansea University/Bristol University: the collaboration between Professor Archer/Dr McCarthy and Professor Wayne McIIlwraith, Dr Lewis Francis, Professor Anthony Hollander will facilitate knowledge transfer between these universities of the progenitor cell work. How will they benefit from this research? The advantages of cartilage progenitor cells over chondrocytes forequine joint repair is that they can divide to produce more cells at a faster rate and maintain their phenotype when extensively grown in the laboratory. Articular cartilage progenitor cells also hold an advantage over the current trend of using bone marrow derived stem cells for tissue repair procedures. In fact, where the latter have been used for cartilage repair procedures, there are a number of reports that these cells terminally differentiate and start to calcify which of course, is detrimental to any cartilage repair procedure. We also have in vitro evidence to suggest that articular cartilage progenitor cells could be considered superior to bone marrow derived stem cells in producing cartilage capable of functional repair. Work carried out in this study would enable such treatment to be optimised to ensure a positive outcome with more consistent results. Commercial potential: Unlike human therapies, there are fewer barriers to entry (less stringent regulatory requirements and clinical trials) for equine treatment and therefore a faster route to market and less competition. Before being used in equine athletes an in vivo trial will be conducted using experimental animals with induced lesions. This will be carried out in collaboration with Prof Wayne McIllwraith in the US. We envisage that this therapy could be used commercially in as little as two years. Spin-out Company Formation Cardiff University has an established partnership with Fusion IP plc. for the development of commercial opportunities into start up companies. Recently stem cells treatments have become available for equine treatments, notably VetCell Ltd which offers treatment for tendon damage. The success of this treatment suggests the market will be receptive to further stem cell treatments. Spin-out company formation would enable recipients to readily gain access to this potentially new and novel treatment for equine cartilage repair.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Regenerative Biology, Stem Cells
Research Priority	Animal Health
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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