We postulate that if bone marrow stromal stem cells or mesenchymal stem cells (MSC) derived from human ESCs can be enriched for osteoprogentior cells the likelihood of engraftment in to new bone after transplantation is going to be high resulting in long term bone repair and new bone formation in patients with bone diseases. Thus, we propose using human embryonic stem cells as a model system to determine whether TWIST expression can be used to enrich MSCs as a novel cell therapy strategy for bone regeneration. TWIST is a Helix-Loop-Helix transcription factor that is known to be a strong inhibitor of osteoblast differentiation. Our laboratory has recently demonstrated that expressing TWIST enriches for osteoblast precursor cells from adult bone tissue in culture. Therefore, allowing TWIST to be overexpressed in human embryonic stem cell derived mesenchymal stem cells (hMSC) under an inducible promoter should allow the enrichment of MSCs ex vivo, and, when turning off TWIST, osteoblast differentiation will be enabled after transplantation. Our laboratory has spent many years isolating and characterizing both mouse and human TWIST in osteoblasts and we are now embarking on new therapeutic applications of this factor in bone diseases. One of the major shortcomings in using bone marrow transplantation for cell therapy in bone diseases is the lack of osteoblast precursor cells. This lack of precursor cell enrichment results in little, if any engraftment of osteoblasts into new bone. Although external factors are presently being used, gene therapy relies on high levels of osteoblast engraftment if the therapy is going to have any phenotypic effect on bone formation, especially with respect to the following bone diseases; osteoporosis, which is prevalent in women over 50; osteogenesis imperfecta (OI) is a serious bone disorder that is usually present at birth as an inherited disease; metastatic cancers such as prostate and breast that can result in cancer induced bone disease leaving the patient in terrible pain for the duration of their life; and for the repair of damage in patients with tooth loss or weakened facial or skeletal bones. We hypothesize that TWIST will have a dramatic effect on stem cell enrichment, and will improve the outcome of hESC cell therapy in humans with bone disease or in need of bone repair. .
Statement of Benefit to California:
This proposed research will benefit the State of California and its citizens in the following ways. First of all, many Californians are "Baby Boomers" who are over 50 and are now suffering from osteoporosis, dental tooth decay, and osteoarthritis resulting hip and knee replacements. Current medicine is deficient in the supply of tissues for bone transplantation. By generating a renewable supply of human MSCs, we hope to circumvent this short supply for future bone tissue regeneration. Secondly, cancer survivors living in California who are in remission are now suffering cancer induced bone disease either from their surgeries or treatment regimens are in need of bone tissue replacement or repairs to relieve them of terrible bone pain. With CIRM funding we now have the ability to use human embryonic stem cells to study the underlying mechanisms of bone formation that will allow us to customize treatments using the hESCs with patients own stem cells to prevent rejection for use in bone replacement and repairs. This proposed project is simply not feasible due to Federal restrictions on the use of hESC. If our hypothesis proves to be correct and mesenchymal stem cells derived from human ESCs can be enriched for osteoprogentior cells, the likelihood of engraftment into new bone after transplantation is going to be high, resulting in long term bone repair and new bone formation in patients with bone diseases. Therefore, Californians receiving hESC cell therapy for bone repairs should be able to remain ambulatory longer, and living more active lives and with less pain. This in turn results in fewer hospital visits for bone fractures. Reduced hospital costs will result in less financial burden on the State of California Health Insurance rates.
SYNOPSIS: In this proposal the investigators wish to differentiate hESCs into MSCs to determine the role of TWIST in hESC/mesoderm differentiation. The hypothesis is that TWIST which interacts with tgf and wnt signaling pathways is a key gene for MSC commitment and self-renewal, allowing increased differentiation to an MSC fate and better expansion of MSCs in vitro; and that this gene will block the differentiation towards osteoprogenitor cells in vitro and in vivo, further substantiating its function to maintain MSCs undifferentiated. Studies are proposed using multiple hESC lines. Parallel studies will be done in adult derived MScs and dental pulp stem cells. SIGNIFICANCE AND INNOVATION: This proposal involves developing methods to generate skeletal cells for therapies of osteoporosis and articular disorders. These are important goals, as with the ageing of the population these diseases are becoming more prevalent. Molecular insights in how to manipulate cell populations to provide robust regeneration of bone / cartilage in vivo are therefore significant. The applicant aims to study the role of a transcription factor, TWIST, in the maintenance of stem cell potency in the human mesenchymal stem cells induced from the human embryonic stem cells. The similar role of TWIST in human bone marrow stromal stem cells and dental pulp stem cells will be studied in parallel. A large population of aging people and patients with inherited diseases are suffering bone diseases such as osteoporosis, osteogenesis imperfecta, and osteoarthritis et al. It is in urgent need to establish model systems in which stem cells are employed as therapeutic sources of bone repair, replacement and regeneration. STRENGTHS: A strength of the proposal is the investigators included in the studies, who have expertise in all aspects of the proposal. The goal of evaluating molecular mechanisms that will enhance the robustness of MSC proliferation and robustness of their capacity to regenerate skeletal tissues in vivo are important. All tools for the proposed studies (inducible TWIST1 construct and a DN construct to mimick TWIST1 null cells) are available. The proposed studies represent a natural extension of the PI’s previous and current research. The originality of the proposal, and inclusion of a strong collaborator Dr. Songtao Shi are also strengths. Many experiments are carefully designed and back-up plans provided. The likelihood of successful completion of the proposed studies is high. WEAKNESSES: Although the investigators indicate that the ESCs are needed to validate the role of TWIST1 during mesoderm/mesnechymal cell commitment in development (last paragraph page 4), none of the studies address this question. If this is not an important goal of the proposal, it is not clear to this reviewer that use of ESCs rather than MSCs derived from bone marrow or dental pulp will aid in the characterization of the role of TWIST to maintain MSCs undifferentiated and block differentiation to osteopropgenitor cells. In general, the proposal is not well written. It is not clear what the PI really wants to do. The proposed studies apparently do not match with what is stated as the overall goals of this proposal (Research Proposal Page 4). It appears that part of the proposal is likely to have been copied-and-pasted from other grant applications, as evidenced by the fact that figures that are referred in text do not exist and numerous unlisted references are cited in the text. In addition, a tetracycline inducible system is proposed in Aim 2c, but an estrogen inducible system will be developed instead in the timeline page. The PI appears to have good training background, and has done pioneering work in the characterization of functional analysis of TWIST in osteoblasts, as she claims. However, the PI is a middle author in 9 of 10 selected peer-reviewed publications that are listed in the PI’s biosketch, which raises a concern. Details are lacking in some places where they are needed, which makes the proposal difficult to follow. However, a pretty detailed quantitative real-time PCR protocol is provided instead. DISCUSSION: A reviewer stated that the aims do not justify the use of hESCs, that the work could be done with mouse ES cells to achieve the same goals. The proposal was regarded as being poorly written, a "cut and paste" job given the lack of attention to relevant details, and was not hypothesis-driven. A reviewer found that the purpose for comparing differentiated mesenchymal cells to bone marrow stromal stem cells was unclear, as was the overall rationale for adding TWIST. Another criticism: the goals did not align with proposed studies. The proposal "lacked real thought".