The overall goal of this proposal is to explore a new stem cell-based treatment for major defects in the orofacial regions resulted from burns, physical injuries, genetic diseases, cancers, infectious diseases, and recently, bisphosphonate-associated osteonecrosis of the jaw (BONJ), using the patient’s own stem cells obtained from the oral cavity known as orofacial mesenchymal stem cells (OMSCs).
The standard surgical reconstruction of orofacial defects relies on different sources of bone grafts harvested from distant anatomical site of the same patient or other donors. However, those approaches are associated with higher morbidity and unpredictable clinical outcomes. Evidences have shown that bone marrow mesenchymal stem cells (BMMSCs) could be a promising alternative for bone reconstruction but not in the orofacial region. These clinical results may be due, in part, to the fact that orofacial and long bones are derived from different cell origins, termed as neural crest cells and mesoderm, respectively. In addition, OMSCs are readily accessible from the oral cavity and can be easily expanded for cell-based therapies due to their inherently high proliferative capability. These evidences suggest that neural crest cell-associated OMSCs might be a superior cell source for orofacial bone regeneration as compared to BMMSCs.
In this study we will compare human OMSCs and BMMSCs in terms of stem cell characteristics and will test their tissue regeneration capacities in the restoration of orofacial defects including the recently drug-induced bone necrosis defects caused by the commonly used drug, bisphosphonate in our established animal models. Our laboratories have recently demonstrated feasibility of using BMMSCs to partially repair craniofacial defects in mouse models. In this proposed study, we will use OMSCs as a model system to determine whether and how individual OMSCs can be utilized as a novel cell therapy for orofacial tissue regeneration. We anticipate that the patient’s own OMSCs will be capable of forming orofacial tissues and will highlight future clinical treatments for orofacial defects.
There is a great clinical demand for developing more optimized approaches to repair facial defects caused by burns, trauma, genetic anomalies, cancers, and recently, the devastating drug-induced osteonecrosis of the jaw associated with the commonly used drug, bisphosphonate (BONJ). Current therapeutic approaches are deficient in supplying appropriate tissues for major facial reconstruction. By generating an optimal supply of human orofacial mesenchymal stem cells (OMSCs) for stem cell-based therapy, we hope to circumvent the limited tissue resource and provide a more superior cell source for future facial tissue regeneration. More importantly, Californians who are head and neck cancer survivors, or suffer esthetic and functionally debilitating orofacial defects will benefit from the advances in stem cell biology and its clinical applications, specifically in the field of orofacial reconstruction. In this proposal, we will expand current knowledge of stem cell biology of OMSC and test the feasibility of utilizing these autologous stem cells in the treatment of diseases such as BONJ. The novel approach in the reconstruction of the orofacial defects using OMSC-based therapy will replace standard paradigm of treatment which involves multiple surgeries, lengthy operating time, cost, and morbidity to the patients. The success of this proposal will not only benefit the people of California, but will have high impact on the state economy by reducing the medical cost and overall financial burden on the State of California Health Insurance.
SYNOPSIS: This study proposes to characterize stem cell properties of newly identified orofacial mesenchymal stem cells (OMSCs), and their use to repair orofacial bone defects. The use of bone marrow mesenchymal stem cells (BMMSCs) for orofacial skeletal repair is suboptimal, due to the distinct properties and embryonic origins of each progenitor SC population. Easily accessible OMSCs are highly proliferative, and provide a more robust stem cell source.
The proposal is organized into three aims:
Aim 1 will determine whether human OMSCs are multipotent postnatal stem cells. Human OMSCs will be isolated and induced in vitro to define their differentiative potential. 10 single colony-derived OMSCs will be isolated and characterized using histological, IHC, molecular and FACS analyses. Their differentiative potential – osteogenic, adipogenic, chondrogenic – will be defined. Known BMMSC markers will be used to characterize OMSCs, including STRO-1, CD18, CD146, CD73, CD90, SSEA4, OCT4, and ALP, by FACS. Cell surface signature will be linked to differentiative potential. 20 single cell colonies will initially be isolated, and 10 lines demonstrated to proliferate over 50 population doublings will be used in the proposed characterizations.
Aim 2 will define the roles of Bmp-2 and bFGF in human OMSC differentiation. Human OMSCs will be isolated by FACS. Bmp-2 and Fgf signaling will be modulated via viral infection, siRNA, and dominant negative (DN) receptor construct transfection, to affect osteogenic induction. Based on preliminary data demonstrating that Bmp2 is down-regulated, and Fgf was up-regulated in OMSCs relative to BMMSCs, the expression of these growth factors will be modulated, and used to assess functions in OMSC proliferation and differentiation. Fgf and Bmp2 expressing OMSCs and HA/TCP carrier will be transplanted subcutaneously into immunocompromised mice, and analyzed at 8-12 weeks for mineralized tissue formation. siRNA will be used to down-regulate Fgf and Bmp2 signaling in OMSCs, and the effect on stem cell maintenance and/or differentiation will be assessed.
Aim 3 will test the feasibility of reconstructing orofacial defects including bisphosphonate-associated osteonecrosis of the jaw (BONJ), using OMSCs in a swine animal model. Swine OMSCs will be isolated and characterized, and compared to human OMSCs. Swine OMSCs will be used to repair orofacial, critical sized bone defects in the BONJ swine model.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal is built on the premise that orofacial derived bone marrow mesenchymal stem cells (OMSCs) are different that BMMSCs derived from axial or appendial (long) bones and are better suited for repair of craniofacial bones. The proposed studies are significant and innovative, in that they will explore a new stem cell based treatment for repairing major defects in the orofacial region, using an autologous cell based therapy. This is a highly significant clinical problem. The PI’s development of the swine model is original, and clinically relevant to humans, based on similar size, shape, and morphology. The properties and regenerative capacity of human OMSCs and BMMSCs will be compared. The proposed studies have the potential to significantly move the field forward, and to improve craniofacial repair therapies, by using NCC derived progenitor cell populations.
The strengths of this application include the expertise of the PI and his collaborators in MSCs and orofacial surgery, the seeming differences between OMSCs and BMMSCs including their neural crest origin, the focus on human MSCs, and the preliminary establishment of a large animal model for bone regeneration. There is preliminary data which demonstrates that: a) Fgf and Bmp2 are differentially expressed in OMSCs and BMMSCs; b) Bmp signaling is involved in OMSC differentiation; c) Generation of the biphosphonate associated osteonecrosis of the jaw (BONJ) mouse model; d) preliminary characterization of the BONJ swine model.
These strengths are balanced by some conerns about the scientific plan and feasibility.
The exact methods used to isolate OMSCs, from what orofacial structures, is not provided, although experimental approaches used to manipulate these stem cells, anticipated results, and alternative strategies are discussed. One reviewer felt that there is no evidence that OMSCs are stem cells. They might very well be progenitor cells. Other reviewers recognized difficulties in characterization of the desired cells. Some of the preliminary data are less convincing: the immunohistochemistry shows almost complete overlap of the cell surface molecules FGFR1,- 2 and BMPR1 with the nuclear stain DAPI, so differences in the MSCs as well as the likely heterogeneity of the isolated clones is hard to determine. Heterogeneity of the cells within an isolation may not yield differences in bone restoration but are likely to confound understanding of the mechanisms.
Finally, concerns were raised over the choice of the swine model for the reconstruction experiments. It is unclear whether swine and human OMSCs are similar enough in their cell surface marker signatures to ensure that demonstrated repair methods in swine will be directly relevant to human repair approaches. The lack of demonstrated ability to identify human and swine OMSC lines that can be passaged for 50 population doublings, while remaining in an undifferentiated state, is also a concern. Finally, if a model already exists in the mouse why use the swine system? Advantages of a large animal model should be balanced with the ease of manipulation and availability of a tremendous amount of genetic background.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Shi is a talented investigator with many important contributions to his field, as shown by his list of publications. He is already recognized as an established leader in the field of craniofacial stem cell characterizations, based on seminar publications and numerous invited presentations. I have no hesitation affirming that he and his team are perfectly qualified to perform all the specific aims of this grant.
Dr. Shi trained as a dentist in Beijing and received a PhD in craniofacial biology at USC in 1994 with three years of postdocatoral training in skeletal biology at UCSF. After a year in private practice, he was a clinical fellow in craniofacial and skeletal diseases at NIH for 4 years. After 3 years as Section Chief of the Dental Biology Section in the same NIH Branch, he became assistant professor in 2006 at USC School of Dentistry.
He has a number of papers in both specialty and high profile journals on mesenchymal stem cells. He has an NIH grant on stem cells from human exfoliated deciduous teeth and a pending NIH grant on applying dental stem cells in the swine model (which he states has no overlap with this application).
The career development plan of the PI is to “further develop my career in stem cell research to incorporate translational medicine”. His long-term goal is to “be an outstanding clinician-scientist engaged in exploring mechanisms of stem cell-associated diseases and translating stem cell technologies to clinical applications”. He presents a reasonable career development plan with plans for training in clinical trials, translational issues and use of human ESCs (no mention of why this), organized by NIH and other organizations. He also mentions a 2 month stay in Australia to work with a collaborator on a clinical trial. He has initiated lab meetings with Dr. Lee, a clinician-scientist specializing in oral and maxillofacial surgery with expertise in transplantation of MSC in animal models, who will provide exposure to ongoing NCI/NIH supported clinical trials. Training in human bone diseases will be received in monthly consultations with Dr. Wen Lou, and oral pathologist, and Dr. Shi will visit long-term collaborator Dr. Stan Gronthos, who is using human SCs to repair myocardial infarction patients. He provides a list of metrics to judge his career development.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: USC School of Dentistry has committed over 12000 sq ft laboratory space, a competitive salary and start up package, and excellent fringe benefits. An internal advisory committee has been established, to review overall progress and provide advice on research and future directions.
There is a strong letter from Dr. Slavkin, the Dean of the Dental School outlining how Dr Shi fits into the plans of more tissue regeneration in the dental school as well as at USC. There is a commitment for space, salary and protected time as well as other efforts to promote Dr. Shi as a leader in the field. USC is the best institution for these studies, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims.
The institutional support at USC appears to be excellent. The institution has established an ES Cell Core laboratory, run by Dr. Martin Pera, Director of the Center for Stem Cell and Regenerative Medicine, providing access and support to all Core scientists. The School of Dentistry has reduced Dr. Shi’s teaching load to 20-30 hours/year, and has provided excellent laboratory space, facilities, and USC infrastructure. Dr. Shi is being promoted as a leader in translation of stem cell research into the clinic, and is provided with annual travel expenses to attend meetings. USCSD has a strong record of promoting the development of new biomedical faculty, and has received 25 Million to establish the Broad Institute for Integrative Biology and Stem Cell research at the Keck School of Medicine of USC, and anticipates the establishment of the USC School of Dentistry Orofacial Regeneration Center, to promote stem cell regeneration of lost of damaged tissues.
DISCUSSION: There was discussion about whether sufficient evidence was presented that the proposed cells are stem cells (i.e., that they can self-renew). Another panelist commented that the RFA embraces stem cells and other progenitor cells, as long as the cells might be clinically relevant. At this point, the panel’s discussion turned to the importance of the proposal as a translational effort for regeneration of orofacial bone, or increased bone formation. This therapy could be important in many clinical situations including: nasopharyngeal cancer, injury, cleft palate, and craniosynostoses. Finally, the panel expressed strong support for the candidate; as a dentist with clinical and basic experience, this person is poised to become a leader in the field.