Improved cartilage regeneration through the selection and use of highly chondrogenic subpopulations of bone marrow mesenchymal stem cells

The objective of this grant is to develop a reliable and predictable stem cell therapy for the treatment of diseases of cartilage. Mesenchymal stem cells (MSCs) from bone marrow can be used to generate chondrocytes (cartilage cells) for direct implantation or for use in tissue engineering protocols that can be used to create new cartilage in the laboratory. However there is a marked variation in the outcome of cartilage formation when using cells from one patient to another. We have shown that there is also marked variation in cartilage formation when using different clonal populations of MSCs from the same patient. By selecting the most chondrogenic and least chondrogenic of these clones we have been able to undertake a comparison of all the genes expressed by MSCs that are more or less able to form cartilage. Through analysis of differentially expressed genes we have identified one that can be used as a cell surface marker of the most chondrogenic MSCs. Its increased level of expression on undifferentiated MSC clones is associated with a higher volume and quality of engineered cartilage after chondrogenic differentiation and tissue engineering on three-dimensional scaffolds. Separation of MSCs into those expressing the marker and those not expressing the marker on their cell surface allows comparison of the chondrogenic capacity of each subpopulation. In this way we have shown that the subpopulation expressing the marker is clearly more chondrogenic (ie better able to form cartilage) than the subpopulation that does not express the marker. Furthermore, measurement of specific collagens has provided data suggesting that the increased cartilage formation is not associated with an increased risk of calcification of the new cartilage (this is usually an inherent problem when using bone marrow stem cells to make cartilage). These novel observations give us the unique opportunity to develop a methodology for the production of MSCs that are predictable in their improved capacity to form cartilage.

This selected population of cells could be used in cartilage tissue engineering procedures or they could be implanted directly into cartilage lesions without the need for in vitro tissue formation. We now wish to develop robust methods for isolation of stem cells expressing the new marker so that we can develop a cell production method that in a Good Manufacturing Practice (GMP) setting that will be required for regulatory reasons if the cells are to be used to treat patients. We then wish to test this population in a sheep model of articular cartilage damage. We also wish to investigate the capacity of cells expressing the marker to suppress immune responses. Bone marrow mesenchymal stem cells are normally able to suppress some aspects of the immune response but we do not yet know if this property is retained in those cells expressing the new marker. This information may be critical in deciding whether in future to develop strategies for cartilage repair based on the patietns own cells or donated cells. Finally we wish to establish if the marker is a passive molecule that happens to be related to cartilage formation or if it plays a mechanistic role in the way stem cells form cartilage.

We have identified the inducible tyrosine kinase-like orphan receptor 2 (iROR2) as a cell surface marker of the most chondrogenic mesenchymal stem cells (MSCs). Its increased level of expression on undifferentiated MSC clones is associated with a higher volume and quality of engineered cartilage after chondrogenic differentiation and tissue engineering on three-dimensional scaffolds. We now want to exploit this discovery for future clinical use.

There will be three strands of work exploring a) the feasibility of producing a therapeutic based on iROR2+ve selection of MSCs b) the immunological properties of iROR2+ve and iROR2-ve cells and c) the mechanism of iROR2 enhancement of chondrogenesis. Feasibility of producing a therapeutic will be investigated by developing a FACS or magnetic cell sorting (MACS) method that is reliable and leads to the production of predictably more chondrogenic cells. Once a production method has been established using human cells we will then investigate if immunosuppression is needed for the xenogeneic implantation of human MSCs into sheep or if the immune privilege of the implant site and the immunoregulatory properties of MSCs will make immunosuppression unnecessary. A pivotal sheep study will then test the efficacy of human iROR2+ve cells in a sheep articular cartilage repair model compared with controls. We will use a new sheep model in which mature tissue engineered cartilage is integrated with the host natural cartilage using our "Cell Bandage" technology. Immunological properties of iROR2+ve and -ve MSCs will be investigated using standard in vitro T cell proliferation assays. The role of ROR2 in MSC chondrogenic differentiation will be investigated by knock-down of the ROR2 gene and by its upregulation in vitro. Zinc-finger technology will be used to ensure stable expression of the full-length gene or the antisense sequences, with evaluation of chondrogenesis in our cartilage tissue engineering ass

The PI and co-PIs all have extensive experience of translating laboratory research into clinically relevant protocols. The PI was a key member of the team that produced the first tissue engineered trachea, that was implanted in a patient in Spain, successfully treating her life-threatening bronchomalacia. That project was possible because of the PIs original work on MSC chondrogenesis for osteoarthritis that was adapted for the engineering of tracheal cartilage. The PI and Dr. Kafienah have jointly developed a "Cell Bandage" technology that is currently being tested in patients with meniscal tear. The original laboratory work was funded by Arthritis Research Uk and then the translational work was supported first by a Wellcome Trust University Translation Award and then by the Technology Strategy Board, leading to the formation of a spin-out company, Azellon Cell Therapeutics that is overseeing the on-going trial and intends to take the therapy through product development. The PI is Chief Scientific Officer of Azellon and closely involved with the translational work. Professor Goodship was part of the team developing a stem cell therapy for the treatment of ligament damage in horses, exploited through the formation of VetCell, which continues to market this therapy and is looking to extend to treating human patients in the future. Professor Wraith has taken his fundamental immunology research through translational exploitation via his spin-out company, Apitope, that is currently running clinical trial of oral peptides for the treatment of multiple sclerosis and other autoimmune diseases.

This collective experience in driving the translational agenda will give us a huge advantage in exploiting iROR2+ve cells in cartilage repair therapies, both through establishing clinical trials and when appropriate through commercial exploitation. A patent on these cells has been filed and the University of Bristol will continue to develop the patent through National phase in parallel with the academic studies described in this project proposal.

Our intention is to progress to clinical trial in traumatic cartilage injury and early osteoarthritis. This may initially require academic funding and collaboration with orthopaedic surgeons in Bristol. But ultimately scale-up will be through commercial exploitation with an appropriate partner. We are already building links with potential future partners and we will strengthen these as the data from our proposed pre-clinical studies become available so that by the time we are ready for a Phase IIa clinical trial we may already be in a position for some commercial involvement and this will grow as we move to Phase IIb studies and beyond.

In his role as President of the International Cartilage Repair Society the PI is in contact with a very wide range of commercial partners with an interest in regenerative medicine and this will enable him to maximise the range of options for this project to be exploited.

Therefore we will generate a high level of long-term impact from these studies through the clinical and commercial routes described here as well as the communication strategy described above.