Combining iPSC and gene editing with cell therapy to develop the next generation of MSC-based therapeutics to counteract bone fragility in OI.

Bone loss and skeletal fragility impose a huge healthcare and economic burden, affecting millions of people in the world due to the ageing process, microgravity, prolonged immobilisation, or pathologies such as osteoporosis or the brittle bone disease osteogenesis imperfecta (OI). OI develops because a genetic defect causes the main component of the bone matrix (type I collagen) to be faulty, preventing the bone-forming cells (osteoblasts) from maturing, which causes the bones to become brittle. There is no cure for OI and patients need care all their lives. Thus, developing therapeutics to counteract bone fragility is of paramount importance.

Using a pre-clinical model of OI, we have recently shown that transplantation of human mesenchymal stem cells (MSC) isolated from the pregnancy fluid surrounding healthy babies improved the quality of fragile bones, stimulated the maturation of resident osteoblasts and reduced the activity of resident bone resorbing cells (osteoclasts). However, there are two main hurdles to overcome before MSC can be routinely used in the clinic. First, MSC isolated from donor organs or pregnancy fluid progressively lose their regenerative potential whilst they are cultivated in the laboratory to reach sufficient numbers. Second, we need to understand how MSC work in vivo to improve transplantation protocols and develop tests to determine the repair potential of putative new donor cell samples. Luckily, a new source of MSC is rejuvenated cells, also called induced pluripotent stem cells (iPSC). Skin cells, which can be isolated from healthy or OI patients, can be rejuvenated in vitro and the genetic defect causing OI can be repaired through genome editing. Since iPSC do not change during culture, it is possible to obtain sufficient number of cells before converting them back into MSC. These cells are called induced MSC (iMSC). This raises the possibility to develop the next generation of cell therapy using iMSC derived from the patient's own cells.

The aim of this project is to to test the potential of iMSC to counteract bone fragility and replace the use of primary MSC and also to understand how donor MSC ameliorate OI bones. This project will test whether transplantation of iMSC (derived from healthy iPSC or from OI-iPSC following correction of the genetic defect) into a pre-clinical model of OI improves bone quality and normalise osteoblast function. We will also establish whether the differentiation of donor MSC towards the osteoblast lineage directly contributes to bone formation and/or whether the tiny sacks (called exosomes) released by MSC function as activators to stimulate resident osteoblasts and/or inhibit osteoclasts. Finally, we will use ex vivo co-culture to assess how the OI genetic defect impedes the behaviour of OI human and mouse osteoblasts and determine whether the function of bone-forming and bone-resorbing cells can be modulated by human MSC through direct cell contact, or via exosomes or the soluble proteins secreted by human MSC.

Our long-term goal is to develop personalized treatments for people suffering from bone loss and at risk of fractures using patients' own skin cells.

Osteogenesis imperfecta (OI) is characterized by skeletal fragility and compromised osteoblast function. Transplantation of human mesenchymal stem cells (MSC) isolated from amniotic fluid (AFSC) in OI mice improved bone quality, stimulated osteoblast maturation and decreased osteoclast activity. However, primary MSC lose their regenerative potential during in vitro expansion, which raises the issue of standardisation and donor cell stability.

An alternative replicable source of human MSC is induced pluripotent stem cells-derived MSC (iMSC). In this project, we propose to test the efficacy of human iMSC to counteract OI bone fragility and also to determine secondary solutions for the treatment of OI (transplantation of iMSC-derived osteoblasts or exosomes or soluble proteins released by iMSC). Our first objective will evaluate and compare the efficacy of iMSC (derived from healthy fibroblast-iPSC, AFSC-iPSC and OI-iPSC following correction of the OI mutation) and AFSC to improve bone mechanical and structural properties following transplantation in oim (recessive) and G610C (dominant) OI neonate mice. We shall next evaluate the in vivo efficacy of AFSC-derived pre-osteoblasts, AFSC-exosomes and AFSC-soluble proteins. Finally, we shall use ex vivo cocultures, imaging and qRT-PCR to investigate the impact of OI mutation on the functionality of human and mouse osteoblasts (i) and determine whether osteoblast and osteoclast functions are modulated by MSC, either through direct cell contact, or via the release of exosomes or soluble proteins (ii).

Our long-term goal is to develop the next generation of iMSC-based personalized therapy using patients' own iMSC, their osteoblast-differentiated derivatives or their by-products, to counteract bone loss and ameliorate the quality of life of people affected by skeletal fragility.

Babies born with OI are affected throughout their lifetime. The costs of managing the disease and caring for patients by healthcare providers and society are extremely high. A study [Public health genomics. 2011;14(3):153-61.] demonstrated that each hospital admission of an OI patient cost approximately £3,000, and each patient (50,000 individuals in Europe) is admitted on average twice a year. As discussed in the case for support, there are no curative treatments for OI. The annual cost savings associated with treatment of patients with severe forms of OI across the EU would be more than £40 million over 5 years after introduction of stem cell therapy.

Other bone pathologies also take a huge toll economically and have a devastating impact on the population, affecting individuals and their families from a pediatric to a geriatric age. Worldwide, osteoporosis causes more than 8.9 million fractures annually. In the UK, there are 230,000 osteoporotic fractures per year, which cost about £2.1 billion per year. The direct (i.e. hospitalization and nursing care) and indirect (i.e. loss of productivity) cost of caring for fractures is very high. Bone diseases can result in physical and mental health and in some cases, in death. Fractures affect the functional status of sufferers and increase the risk of other complications such as pressure sores, pneumonia, and urinary tract infection. They also lead to psychological consequences, as a result of a decreased quality of life.

Our work on OI will provide important knowledge and has considerable translational and commercialisation impact.

Specific beneficiaries:

For patients: Patients with fragile bone disorders will directly gain from the development of new therapeutic strategies to increase bone strength and quality and decrease bone pain.

For researchers: Our findings will expand the respective fields of stem cell biology, molecular biology, biomechanics, cell therapy and bone metabolism, providing proof-of-principle for the use of human iPSC-derived iMSC to overcome two of the major hurdles of primary MSC therapy.

For clinicians: As stem cell transplantation for OI moves into the clinic (BOOSTB4 phase I/II clinical trial), it is important to understand the mechanisms by which donor MSC exert their beneficial effects to further develop innovative stem cell-derived treatments and to investigate the bone regenerative potential of alternative replicable and standardized source of MSC to replace the use of primary cells. It is likely to be of interest to pediatric as well as geriatric orthopedic surgeons and physicians, for the purpose of bone repair and regeneration, and to restore osteoblastic depression.

The benefits of this research are not restricted to the scientific community. The knowledge gained will also be of benefit to industry and the commercial private sector. For example, the banking of human iPSC-derived iMS, and the development of cell-free therapeutics to promote bone health.

We also anticipate that this project will lead to preclinical testing of putative new samples for cell therapy and hence, the commercialization of assays to test the in vitro bone regenerative potential of cell samples. This will benefit the UK economy either through the creation of new spin-out companies via UCL Innovation, or by adding values to existing large pharmaceutical companies through license or sale of intellectual property rights.