Bone or Cartilage Disease

Coding Dimension ID: 
279
Coding Dimension path name: 
Bone or Cartilage Disease
Funding Type: 
Early Translational II
Grant Number: 
TR2-01821
Investigator: 
Name: 
Type: 
Co-PI
ICOC Funds Committed: 
$5 391 560
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Other
oldStatus: 
Active
Public Abstract: 
Like most tissues of the body, bone possesses a natural regenerative system aimed at restoring cells and tissues lost to natural cell aging, disease or injury. These natural regenerative systems are complex combinations of cell growth factors and support structures that guide and control the development of specialized bone stem cells. However, the regeneration process may still fail, for multiple reasons. For instance, the degree of skeletal injury may be so great that it overwhelms the natural regenerative capacity. Alternatively, the natural regenerative capacity may be defective; this is exemplified by osteoporosis, a frequent condition affecting post-menopausal females and elderly males and females. Osteoporotic individuals have severe declines in stem cell numbers (10-fold decrease from age 30 to 80) and stem cell function (tendency of stem cells to turn into fat rather than bone cells with age), leading to bone loss and “fragility fractures” that typically would not occur in persons with normal stem cell number and function. Thus, there is a tremendous need for therapies to increase the number and function of endogenous adult stem cells with the potential to build new bone. One option is to introduce so called mesenchymal stem cells (MSC) from the patient to bone repair sites. However, significant hurdles to autologous MSC use include the need for 2-3 week culture times to isolate MSC before application. Moreover culturing introduces infectious and immunogenic risks from prolonged exposure to animal products and cancerous risks from cellular gene changes in culture. In addition, once isolated, MSC require appropriate growth factor stimulation to form bone. Finally, MSC isolated from patient tissues such as fat or bone marrow are heterogenous and of undetermined composition—making growth factor dosing and conformance with FDA regulations for defining target product identity, purity, and potency more difficult. To circumvent these problems, we have identified and purified the cells at the origin of human MSC. We have termed these perivascular stem cells (PSC) because they are natively localized around all arteries and veins, forming the key cellular component of the natural regenerative system. In a significant breakthrough, we are able to isolate these cells within hours from adipose tissues in sufficient numbers for therapy without the need for culture. This realizes the possibility of harvesting and implanting stem cells during the same operative period. In another breakthrough, we have identified a potent growth factor NELL-1 that potently amplifies the ability of PSC to form bone and vascular structures. This has led to the development of our target PSC+NELL-1 product, which effectively stimulates and augments the body’s natural bone regenerative system by providing all the components (stem cells, growth factor, and allograft bone support structure) necessary to “jump start” as well as maintain the function of bone stem cells.
Statement of Benefit to California: 
This FDA oriented proposal focuses on crucial preparatory work required before IND-enabling preclinical studies on our Developmental Candidate for bone formation and regeneration. Our Developmental Candidate provides a complete package of stem cells, bone growth factors, and scaffold to build an optimized microenvironment to “jump start” bone formation in normal and impaired bone healing conditions. We have generated very promising preclinical data on our Developmental Candidate’s superior bone formation and regeneration efficacy. In addition to its significant impact on health care, this highly multi-disciplinary project by our team has many near-term and long-term benefits to the State of California. 1. Besides direct health costs, musculoskeletal injuries and diseases are the leading cause of work-related and physical disability in the United States. Hard working Californians are responsible for California’s annual gross domestic product of $1.8 trillion, which rank our state among the top eight largest economies in the world. By promoting the repair of both normal and healing-impaired bone in a safe and effective manner, our mature technology will reduce the loss of work productivity at the front end, reduce work disability costs, and reduce the loss of state income tax. 2. Local osteogenic stem cells decline with age (from 1/10,000 in newborns to 1/250,000 by age 30 and 1/2,000,000 by age 80), leading to osteoporosis, poor bone quality, and fragility fractures. In 1998, the health care burden for osteoporosis exceeded $2.4 billion in California alone. A whopping 64% of the $2.4 billion was caused by hip fracture. If our Development Candidate is successful in healing existing fractures in impaired bone, it may also translate to therapies to prevent fractures in impaired bone. This will significantly reduce the long-term health care burden for California’s public health insurance program. 3. This project directly adds jobs at [REDACTED] and at the California-based companies that are involved in this project. 4. This project will produce intellectual property that is owned by t [REDACTED]. Our team has a track record of attracting out of state private investment to invest in California and of procuring supplies and equipment from strategic California-based companies. 5. This mature project is precisely the type of cutting-edge, multi-disciplinary stem cell project that Californians imagined when they approved proposition 71 in 2004. The establishment of CIRM has transformed the research infrastructure at [REDACTED], increased our ability to recruit world class stem cell scientists, and attracted the attention of superb scientists from other disciplines to this new field. Working together, our team has compiled an impressive list of accomplishments and we are confident in our abilities to take this project to IND submission in a timely fashion. Funding of this project will fulfill the promise of proposition 71.
Progress Report: 
  • Background: unsuccessful bone repair
  • Most organs can regenerate cells lost to ageing, exposure to adverse conditions, ephemeral lifespan, disease or injury. Regenerative systems are complex combinations of growth factors and anchorage molecules which support, guide and control the maturation of specialized stem cells. The regenerative process may still fail, for multiple reasons. Bone fractures will sometimes not heal spontaneously because parts of the broken bone do not join anymore, or because bone regeneration itself is impaired, like in osteoporosis, a frequent condition that affects post-menopause females and elderly people of both genders.
  • A novel class of bone-healing stem cells
  • Since specialized bone-forming cells have not been identified, which stem cells could be used for the cell therapy of bone fractures? We have identified and purified novel stem cells, localized around blood vessels and therefore named perivascular stem cells (PSCs). We have also demonstrated that Nell-1, a potent osteogenic growth factor we have developed, efficiently turns PSCs into bone cells. In 2011, we have been granted support by the California Institute for Regenerative Medicine to develop a product to heal critically fractured bones in osteoporotic – or not – patients or to perform spine fusion in order to correct skeletal defects. PSCs will be purified from the patient’s own fat tissue, a well-documented, rich source of these cells, and embedded in a biocompatible scaffold in the presence of the bone forming growth factor. The resulting compound, inserted at the site of the fracture, will provide bone-forming stem cells 1-precisely characterized in terms of origin and identity; 2- derived from the patient, hence not rejected; 3- not cultured, therefore similar to their natural counterparts.
  • Progress after one year of research supported by CIRM
  • We are now close to the end of the first year of our CIRM supported studies. In this initial period we have, first, validated the stem cells of use with regard to stringent biologic criteria: how many of these cells can be purified from fat tissue? How viable and pure are these cells after the sorting process? Do stem cell numbers and quality depend on the age, sex and corpulence of the donor? Following analysis of about 80 distinct fat harvests (lipoaspirates) we have determined that robust bone forming PSC can be reliably purified, in sufficient numbers, from male and female donors in all age and weight ranges. We have also shown that PSC are at least as good, in terms of bone forming potential, than conventional mesenchymal stem cells (the candidate stem cells, so far, to be used for cell therapies of bone defects amd injuries), while being significantly superior regarding purity and safety.
  • Transplantation experiments in mice and rats have revealed that the combination of PSC and the Nell-1 growth factor ensures the most efficient bone regeneration, but that PSC do not give rise to tumors, an important verification in any protocol involving transplantation. These experiments have also demonstrated that PSC stimulate the formation of new blood vessels, an essential requisite for efficient wound healing and tissue repair.
  • Finally, we have embarked on the development of a larger animal model of bone regeneration, that will be used in the second and third years of the projects.
  • In summary, we have reached all the milestones and met the deadlines planned in the original project. The next year will see the further validation of the protocol and the beginning of the development of a stem cell/scaffold/growth factor combination product in the perspective of trials in human patients.
  • Our research group has identified in the human body a novel class of stem cells endowed with strong osteogenic potential, which means capacity to produce bone. These stem cells are attached to the outer aspect of blood vessels, and therefore present in all organs. Our ultimate goal is to use these stem cells in human beings, to perform for instance spine fusion, a procedure used to treat spine deformities (scoliosis) or faulty vertebrae, or to heal complicated bone fractures, especially in patients suffering from osteoporosis, a condition where spontaneous bone repair is compromised. We shall harvest stem cells from the patient’s own abdominal adipose (fat) tissue, a rich source of these cells which is also easy and safe to collect. Stem cells will be purified on an automated machine, named fluorescence activated cell sorter, and immobilized on an engineered biocompatible scaffold in the presence of a novel osteogenic growth factor, that is a sort of hormone which stimulates stem cell development into bone. We have won an Early Translational grant (2011-2014) from the California Institute for Regenerative Medicine to develop this project to the stage where the first clinical trials can be engaged into. In the first year, we had fully validated the stem cell population of use on a large number of human adipose samples and clearly documented the availability, efficiency and safety of these cells independently of the age, sex and weight of the donor. We had also initiated experiments in mice and rats which have been pursued during the second year, finally demonstrating unequivocally the outstanding bone forming ability of these cells in the presence of the appropriate scaffold and osteogenic hormone. We have also, during this 2nd year, developed a large animal model of stem cell mediated bone regeneration which will be indispensable prior to the inception of human treatments. Finally, we have refined the physicochemical properties of the scaffold for optimal osteogenic factor availability and bone growth. In conclusion, all milestones and deadlines included in this grant have been met and our team is steadily progressing toward the medical “translation” of these novel stem cells.
  • Our research group has identified in the human body a novel class of stem cells endowed with strong osteogenic potential, which means capacity to produce bone. These stem cells are attached to the outer aspect of blood vessels, and therefore present in all organs. Our ultimate goal is to use these stem cells in human beings, to perform for instance spine fusion, a procedure used to treat spine deformities (scoliosis) or faulty vertebrae, or to heal complicated bone fractures, especially in patients suffering from osteoporosis, a condition where spontaneous bone repair is compromised. We shall harvest stem cells from the patient’s own abdominal adipose (fat) tissue, a rich source of these cells which is also easy and safe to collect. Stem cells will be purified on an automated machine, named fluorescence activated cell sorter, and immobilized on an engineered biocompatible scaffold in the presence of a novel osteogenic growth factor, that is a sort of hormone which stimulates stem cell development into bone. We have won an Early Translational grant (2011-2014) from the California Institute for Regenerative Medicine to develop this project to the stage where the first clinical trials can be engaged into. In the first year, we had fully validated the stem cell population of use on a large number of human adipose samples and clearly documented the availability, efficiency and safety of these cells independently of the age, sex and weight of the donor. We had also initiated experiments in mice and rats which have been pursued during the second year, finally demonstrating unequivocally the outstanding bone forming ability of these cells in the presence of the appropriate scaffold and osteogenic hormone. We have also, during this 2nd year, developed a large animal model of stem cell mediated bone regeneration which will be indispensable prior to the inception of human treatments. Finally, we have refined the physicochemical properties of the scaffold for optimal osteogenic factor availability and bone growth. In conclusion, all milestones and deadlines included in this grant have been met and our team is steadily progressing toward the medical “translation” of these novel stem cells.
Funding Type: 
Early Translational II
Grant Number: 
TR2-01780
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 927 698
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Other
oldStatus: 
Active
Public Abstract: 
Vertebral compression fractures are the most common fractures associated with osteoporosis. Approximately 700,000 osteoporosis-related vertebral compression fractures (OVCFs) occur each year in the US. Currently, treatment is focused primarily on prevention. When fractures occur in patients with osteoporosis, treatment options are limited because open surgery with implants often fails. Recently, new therapies involving injection of cement into the vertebral body were developed. Unfortunately, these procedures do not regenerate bone tissue, but do incur risks of leakage and emboli. Hence, we need new treatments that directly address both the underlying cause of OVCFs (bone loss) and the inadequate repair mechanisms when fractures occur. We propose to develop a therapy that exploits mesenchymal stem cells (MSCs) stimulated in vivo with PTH (parathyroid hormone) to accelerate bone repair. PTH alone can accelerate fracture repair in healthy animals by activating bone marrow MSCs. However, osteoporotic patients have either decreased numbers of MSCs, dysfunctional MSCs, or both. In these patients, injection of MSCs combined with a PTH regimen could be an effective therapy for the treatment of multiple fractures. Our preliminary data in a mouse model demonstrated that this combined treatment enhances MSC homing to long-bone fracture sites and leads to increased repair. Here, we will build upon this foundation and ask whether a similar strategy is also effective in OVCFs. We hypothesize that PTH administration will lead to increased homing of MSCs to sites of bone fracture. We further hypothesize that PTH promotes the differentiation of MSCs into osteoblasts. Hence, our objective in the proposed study is to determine the effect of injection of MSCs combined with PTH therapy on bone regeneration in a multiple vertebral bone defect model in osteoporotic rats. The optimal doses of PTH and numbers of MSCs per injection also will be determined. Human bone marrow-derived MSCs will be injected into osteoporotic athymic rats with multiple lumbar vertebral bone defects. MSC homing to bone defects will be monitored using micro- and molecular imaging. Subsequent studies will test increasing dosages of PTH to define the optimal dose for maximal enhancement of MSC homing to a fracture. Bone regeneration will be monitored using micro–CT imaging and biomechanical analyses (to determine structural integrity of newly repaired bone). Subsequent studies will determine whether increasing the number of injected MSCs linearly enhances bone tissue formation. These studies will aid in the creation of an evidence base for future clinical trials that could revolutionize the treatment of vertebral fractures and other complex fractures in patients suffering from osteoporosis.
Statement of Benefit to California: 
Approximately 10 million people in the United States are diagnosed as osteoporotic, while an additional 34 million are classified as having low bone mass. The lifetime incidence of fragility fractures secondary to osteoporosis in females over fifty years of age is approximately 1 in 2, and in males over the age of fifty, is 1 in 4. Osteoporosis-related vertebral compression fractures (OVCFs) are the most common fragility fractures in the United States, accounting for approximately 700,000 injuries per year, twice the rate of hip fractures. Approximately 70,000 OVCFs result in hospitalization each year with an average hospital stay per patient of 8 days. Fragility fractures due to osteoporosis also place a severe financial strain upon the health care industry. Estimates show there were approximately 1.5 million osteoporosis-related fractures in the United States in 2001, the care of which cost about $17 billion. Moreover, as the number of individuals over the age of fifty continues to increase, costs are predicted to rise to an estimated $60 billion a year by the year 2030. OVCFs have previously received limited attention from the spine care community. This oversight may be a result of the perception that OVCFs are benign, self-limited problems or that treatment options are limited. However, it has become clear that OVCFs are associated with significant physiologic and functional impairment, even in patients not presenting for medical evaluation at the time of fracture. Current treatment of osteoporotic patients is mostly focused on prevention of OVCFs. There are a few options of treatment when OVCFs actually occur. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, nonoperative management, including medications and bracing, is usually recommended for the vast majority of patients. Unfortunately, large numbers of patients report intractable pain and inability to return to activities. Currently there is no efficient biological solution for the treatment of OVCFs. The proposed study will further develop a biological therapeutic solution that will accelerate repair of OVCFs. The treatment will rely upon a combination of drug and adult stem cell therapy; both are either approved for clinical use or in clinical trials. It will also involve a simple intravenous injection instead of a percutaneous injection of a polymer, which does not restore lost bone tissue. Data generated form this study could potentially revolutionize the treatment of vertebral fractures and other complex fractures in patients suffering from osteoporosis, and so benefit the citizens of California by reducing hospitalization periods, operative costs and loss of workdays, and by improving quality of life for Californians with osteoporosis that are at risk for OVCFs.
Progress Report: 
  • The goal of the proposed study is to develop a treatment for accelerating multiple vertebral fracture repairs. Approximately 10 million people in the United States are diagnosed as osteoporotic, while an additional 34 million are classified as having low bone mass. The lifetime incidence of fragility fractures secondary to osteoporosis in females over the age of fifty years of age is approximately 1 in 2, and in males over the age of fifty, is 1 in 4. Vertebral compression fractures (VCFs) are the most common fragility fractures in the United States, accounting for approximately 700,000 injuries per year, twice the rate of hip fractures. Approximately 70,000 VCFs result in hospitalization each year with an average hospital stay per patient of 8 days. Current treatment of osteoporotic patients is mostly focused on prevention of VCFs mainly using new medicines such as Alendronate and Parathyroid Hormone (1-34). But there are a few options of treatment when VCFs actually occur. New, non-biological, methods have been developed to regain the biomechanical properties of a fractured vertebral body. These methods include the minimally invasive procedures of vertebroplasty and balloon tamp reduction. Both procedures involve injection of synthetic nonbiological material that does not resorb and instead remains a permanent foreign-body fixture in the spine. Ultimately, a biological solution that would promote rapid fracture healing and stimulate normal bone production would be the best for osteoporotic patients with vertebral column injuries. Our pilot studies have shown that bone fractures can be treated by intravenous administration of adult stem cells combined with a dose of parathyroid hormone, which is approved by the FDA for use as an anabolic agent in the treatment of severe osteoporosis. Therefore, in the proposed project we will determine the ability of injected stem cells to migrate to a vertebral fracture site. We will also analyze the combined effect of PTH and stem cells on vertebral fracture repair.
  • The goal of the study is to develop a treatment for accelerating multiple vertebral fracture repair. Approximately 10 million people in the United States are diagnosed as osteoporotic, while an additional 34 million are classified as having low bone mass. Vertebral compression fractures (VCFs) are the most common fractures in the United States, accounting for approximately 700,000 injuries per year, twice the rate of hip fractures. Approximately 70,000 VCFs result in hospitalization each year with an average hospital stay per patient of 8 days. Current treatment of osteoporotic patients is mostly focused on prevention of VCFs mainly using new medicines such as Alendronate and Parathyroid Hormone (PTH). But there are no options of treatment when VCFs actually occur either than bed rest and pain medication.
  • Our goal is to induce efficient vertebral fracture repair by a combined treatment of adult stem cells and PTH. During the last year we treated osteoporotic animals (rodents) with human stem cells, isolated from bone marrow. The cells were injected to the blood circulation followed by PTH treatment for three weeks. In order to evaluate whether the injected cells migrated to the region of the spinal fractures, we used fluorescent cells. Using a highly sensitive camera we were able to track the injected cells in the body of the animals over 55 days. Our results showed that more cells targeted the spine region when PTH was given to the animals compared to a control group that did not receive PTH. In addition, we analyzed the effect of the stem cell treatment on the repair of spinal fractures. Using high resolution CT imaging we found that osteoporotic animals treated with stem cells and PTH had significantly more bone fracture repair when compared to untreated animals and to animals treated with PTH or stem cells only.
  • In conclusion, we have generated promising results demonstrating the efficiency of stem cell therapy combined with PTH for the treatment of vertebral fractures. We will further explore this effect in the 3rd year of the project, aiming to promote this therapy further towards clinical use.
  • During the 3rd year of the project we have been able to show that when stem cells are injected to osteoporotic animals, they migrate and home to vertebral fractures. We also showed that treating the animals with an FDA-approved drug, PTH, enhances the homing of the stem cells (i.e. more cells migrate to the site of the fracture). Using microscopy and specific fluorescent dyes we were able to investigate the fate of the injected stem cells after they arrived to the fracture site. Apparently these cells turn into bone-forming cells and participate in the fracture repair. Indeed, when we measured the repair of the vertebral fractures (i.e. amount of new bone that was formed) we found that the combination therapy of stem cells and PTH had a superior effect compared to the therapies that included only stem cells or were left untreated. We are currently evaluating the use of lower doses of PTH that are equivalent to the dose used for prevention of fractures in osteoporosis patients. In summary, we believe that our results so far merit further investigation of the proposed therapy that might prove beneficial of numerous patients suffering from vertebral compression fractures every year.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00572
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$3 253 464
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Progress Report: 
  • The long-term goal of this proposal is to develop stem cell-based treatment for major defects in the orofacial regions. Bisphosphonate related osteonecrosis of the jaw (BRONJ) is a recently described adverse side effect of bisphosphonate therapy, with an estimated 94% of cases reported in the oncologic patients receiving intravenous nitrogen-containing bisphosphonates (BP). Due to the lack of a testable animal model and limited biological tissue specimens, to date, the patho-physiological mechanisms underlying BRONJ remain largely unknown. We have successfully established BRONJ minipig and mouse models treated with oncologic doses of zolendronate (Zometa)/Dexamethasone (Dex) developed BRONJ-like pathological lesions with similar clinical, radiographic, and histological features as described in the human disease. These models will be used to understand mechanism of BRONJ and find appropriate therapeutic approaches for BRONJ.
  • We isolated a new population of stem cells from human orofacial tissue gingiva, a tissue source easily accessible from the oral cavity, namely GMSC, which exhibited clonogenicity, self-renewal, and multipotent differentiation capacities. Most importantly, GMSC were capable of immunomodulatory functions. Cell-based therapy using systemic infusion of GMSC in experimental colitis significantly ameliorated both clinical and histopathological severity of the colonic inflammation, restored the injured gastrointestinal mucosal tissues, reversed diarrhea and weight loss, and suppressed the overall disease activity in mice. The therapeutic effect of hGMSC was mediated, in part, by the suppression of inflammatory infiltrates and inflammatory cytokines/mediators at the colonic sites. GMSC can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and is a promising cell source for cell-based treatment in experimental inflammatory diseases.
  • In collaboration with investigators in Taiwan, we implanted one type of autologous OMSCs (periodontal ligament progenitors, PDLPs) to treat an orofacial infectious bone defect disease periodontitis. We examined the clinical outcome of three autologous PDLP-treated patients in an effort to provide primary knowledge on the effectiveness of this treatment approach and preliminary clinical evidence for randomized controlled trial in the future. Clinical examination indicated that local implantation of PDLPs may provide therapeutic benefit for the periodontal defects. All treated patients showed no adverse effects during the entire course of follow up. This study demonstrated clinical and experimental evidences supporting a potential efficacy and safety of utilizing autologous PDL cells in the treatment of human periodontitis.
  • Human orofacial bone-derived mesenchymal stem cells (OMSCs) showed distinct differentiation traits from mesenchymal stem cells (MSCs) derived from long bones, mouse OMSCs have not been isolated due to technical difficulties, which in turn precludes using mouse models to study orofacial diseases. We developed techniques to isolate mouse OMSCs derived from mandibles and verified their MSC characteristics by single colony formation, multi-lineage differentiation, and in vivo tissue regeneration. Activated T-lymphocytes impaired OMSCs via the Fas/Fas ligand pathway, as occur in long bone MSCs. Furthermore, we found that OMSCs are distinct from long bone MSCs with respect to regulating T-lymphocyte survival and proliferation. Our data suggest that OMSCs are a unique population of MSCs and have a role in systemic immunity.
  • Embryologic development and amalgamations of the complex array of bones and cartilage in the craniofacial region have revealed that the molecular mechanisms controlling skeletogenesis in the orofacial bones are quietly unique and different from in the axial and appendicular bones. The discrepancy in bone development between orofacial bones and long axial/appendicular bones give rises to specific diseases in the orofacial bone region, such as periodontitis, cherubism, and hyperparathyroid jaw tumor syndrome, which only affect the jaw bones. Therefore, it is not surprising to find that human OMSCs are distinct from BMMSCs in terms of differentiation traits and immunoregulation. MSC mediated bone formation involves in both donor and recipient cells, but only recipient cells contribute to marrow element formation. Our study suggests that both OMSCs and host cells contribute to bone formation in vivo.
  • Ex vivo-expanded BMMSCs are capable of suppressing the T-lymphocyte proliferation and activity in vitro, which provides a foundation for using BMMSC transplantation to treat T-cell-associated disorders, such as acute graft-versus-host-disease (GvHD) in mice and humans. In addition, we found activated T-lymphocyte induced apoptosis of BMMSCs through the Fas/FasL pathway. Our data suggest that OVX induced T lymphocyte activation may contribute to OMSC damage. Although T lymphocyte activation in OVX condition is a major factor for promoting osteoclast function and inhibiting osteoblast function, we can’t exclude other factors that may also contribute to OMSC deficiency in OVX mice. The immune-modulatory property is related to a high level NO production induced by IFN via enhanced iNOS expression in BMMSCs. In this report, mouse OMSCs showed a stronger suppressive effect on proliferation of anti-CD3 antibody-activated T cells, but only partially inhibited T cell proliferation by anti-IFN antibody and the iNOS inhibitor, 1400W. These highly immunosuppressive properties of OMSCs may provide an advantage for tissue engineering in the orofacial region. Surprisingly, mouse OMSCs produced larger amounts of NO than mouse BMMSCs, indicating that OMSCs are more responsive to inflammatory cytokine(s)-induced NO production. We also found that OMSCs were capable of keeping naïve splenocytes including T cell survival more effectively than BMMSCs. Therefore, it is necessary to continue elucidating underlying mechanisms of the interplay between OMSCs and immunity using established various mouse models.
  • Bisphosphonates (BPs) have been used for the clinical treatment of bone diseases with increased bone resorption such as osteoporosis and malignant diseases like multiple myeloma or metastasis to the bone. However, there is increasing evidence associate bisphosphonates treatment with osteonecrosis of the jaws. The detail mechanism of bisphosphonate-related osteonecrosis of the jaws (BRONJ) is unclear and it is very difficult to be treated. In present study, we generated large animal model of BRONJ in miniature pig and treated with allogeneic bone marrow mesenchymal stem cell (BMMSCs) transfusion. Of the 9 miniature pigs received BPs treatment and tooth extraction, 6 pigs disclosed BRONJ with exposed bone. The level of CD4+CD25+ T cells, foxp3+ T cells in the peripheral blood was decreased, while the level of γδ T cells and IL-17 were increased. After MSCs infusion, mucosal and bone healing were achieved, changes in immunity recovered. These findings obtained in a clinically relevant large-animal model of BRONJ provide evidence of the connection of BPs treatment and osteonecrosis of the jaw, as well as the immunity-based mechanism of BRONJ.
  • The long-term goal of this proposed study is to explore a new stem cell-based treatment for major defects in the orofacial regions. Bisphosphonate related osteonecrosis of the jaw (BRONJ) is a recently described adverse side effect of bisphosphonate therapy, with an estimated 94% of cases reported in the oncologic patients receiving intravenous nitrogen-containing bisphosphonates (BP). Due to the lack of a testable animal model and limited biological tissue specimens, to date, the patho-physiological mechanisms underlying BRONJ remain largely unknown. Previously we established BRONJ mouse model and found regulatory T cells can prevent BRONJ in mouse model. Recently, we have established BRONJ pre-clinical model in minipigs and confirmed that regulatory T cells and Th17 cells contribute to the occurrence of BRONJ. In order to further characterized cell-based therapy for
  • orofacial defects, we generated radiation-induced jaw bone necrosis model in minipigs and use mesenchymal stem cell (MSC) implantation to cure the necrosis, suggesting a potentiality of using cell-based therapy for jaw bone regeneration.
  • To further understanding mechanism by which MSCs are capable of regenerating orofacial bones, we showed that MSC-based bone regeneration inhibited by recipient T cells via IFN-gamma and TNF-alpha. Local aspirin treatment can block T cell activity and, therefore, improve MSC-based orofacial bone regeneration. Moreover, we demonstrated that ERK signaling pathway controls orofacial MSC-mediated bone regeneration. ERK1 ⁄ 2 inhibitor treatment rescued bFGF-induced osteogenic differentiation deficiency.
  • Finally, we showed that vitamin C treatment improved capacity of orofacial MSC-mediated orofacial bone regeneration in minipigs through up-regulation of telomerase activity.
  • The goal of this grant proposal is to characterize orofacial mesenchymal stem cells and determine the feasibility of reconstructing the orofacial defects caused by a variety of diseases such as osteonecrosis of the jaw using mesenchymal stem cells. Our study focuses on mesenchymal stem cell characterization, disease model generation, and mesenchymal stem cell-based orofacial bone regeneration in large animal model,
  • • We identified that Erk1/2 signaling regulate both MSC-mediated bone regeneration and immunomodulation (manuscript in preparation).
  • • We showed that MSC-based immunotherapy involves in coupling via FasL/Fas to induce T cell apoptosis (Akiyama et al., Cell Stem Cell 2012).
  • • We have established jaw osteoradionecrosis (ORN) pre-clinical model and shown that mesenchymal stem cell-based implantation can cure ORN in minipigs (Xu et al., Cell Transplantation 2012).
  • • We continue to characterize effectiveness of mesenchymal stem cell-based therapy for bisphosphonate-associated osteonecrosis of the jaw (BRONJ) in pre-clinical model (manuscript in preparation) and confirmed some clinical phenotypes in BRONJ patients (Patel et al., Oral Diseases 2012).
  • The purpose of this grant proposal is to characterize orofacial mesenchymal stem cells and determine the feasibility of reconstructing the orofacial defects caused by a variety of diseases such as osteonecrosis of the jaw using mesenchymal stem cells. Our study focuses on mesenchymal stem cell characterization, disease model generation, and mesenchymal stem cell-based tissue regeneration in small and large animal models,
  • • We identified that Erk1/2 respectively regulate MSC-based immunomodulation and osteogenic differentiation.
  • • We showed that MSC-based immunotherapy involves in coupling via FasL/Fas to induce T cell apoptosis.
  • • We have established jaw osteoradionecrosis (ORN) pre-clinical model and shown that mesenchymal stem cell-based implantation can cure ORN in minipigs.
  • • We continue to characterize effectiveness of mesenchymal stem cell-based therapy for bisphosphonate-associated osteonecrosis of the jaw (BRONJ) in pre-clinical model (manuscript in preparation) and confirmed some clinical phenotypes in BRONJ patients.
  • • We found that inflammation environment inhibits MSC-based bone formation via TNF Alpha and IFN-Gamma.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01249
Investigator: 
Name: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$6 762 954
Disease Focus: 
Bone or Cartilage Disease
Stroke
Neurological Disorders
Heart Disease
Neurological Disorders
Skin Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration. Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic. Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries. The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.
Statement of Benefit to California: 
Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B. We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.
Progress Report: 
  • In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
  • Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
  • In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
  • Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
  • In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
  • We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
  • In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.
  • The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.
  • To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.
  • This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.
  • At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.
  • In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.
  • In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.
  • This progress report covers the period between Sep 01 2013 through April 30 2014, and summarizes the work accomplished under ET funding TR101249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for purposes of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians. At the conclusion of Year 2 an external review panel was assembled and charged with the selection of a single lead indication for further development. The scientific advisory board unanimously selected skeletal repair as the lead indication.
  • In year 3 we accrued addition scientific evidence, using both large and small animal models, demonstrating that a WNT protein therapeutic accelerated bone healing. Also, we developed new methods to streamline the purification of WNT proteins, and improved our method of packaging of the WNT protein into liposomal vesicles (e.g., L-WNT3A) for in vivo use.
  • In year 4 we clarified the mechanism of action of L-WNT3A, by demonstrating that it activates endogenous stem cells and therefore leads to accelerated bone healing. We also continued our development studies, by identifying a therapeutic dose range for L-WNT3A, as well as a route and method of delivery that is both effective and safe. We initiated preliminary safety studies to identify potential risks, and compared the effects of L-WNT3A with other, commercially available bone growth factors.
  • In year 5 we initiated two new preclinical studies aimed at demonstrating the disease-modifying activity of L-WNT3A in spinal fusion and osteonecrosis. These two new indications were chosen by a CIRM review panel because they represent an unmet need in California and the nation. We also initiated development of a scalable manufacturing and formulation process for both the WNT3A protein and L-WNT3A formulation. These two milestones were emphasized by the CIRM review panel to represent major challenges to commercialization of L-WNT3A; consequently, accomplishment of these milestones is a critical yardstick by which progress towards an IND filing can be assessed.
  • With regards to objective 1, we employed established animal models of spinal fusion and osteonecrosis to demonstrate the disease-mitigating activity of our Developmental Candidate, autograftWNT.
  • With regards to objective 2, the mechanism of L-WNT3A action has been demonstrated in ex vivo, non-GLP pharmacology studies.
  • With regards to objective 3, we have completed characterization of the non-GLP substance, WNT3A drug product, and the drug product, L-WNT3A. Methods for reproducible and scaleable research grade production of the drug substance WNT3A, and the drug product, L-WNT3A, have been developed (see below for details). A serum free process has been achieved.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01216
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$3 118 431
Disease Focus: 
Arthritis
Bone or Cartilage Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Arthritis is the result of degeneration of cartilage (the tissue lining the joints) and leads to pain and limitation of function. Arthritis and other rheumatic diseases are among the most common of all health conditions and are the number one cause of disability in the United States. The annual economic impact of arthritis in the U.S. is estimated at over $120 billion, representing more than 2% of the gross domestic product. The prevalence of arthritic conditions is also expected to increase as the population increases and ages in the coming decades. Current treatment options for osteoarthritis is limited to pain reduction and joint replacement surgery. Stem cells have tremendous potential for treating disease and replacing or regenerating the diseased tissue. This grant proposal will be valuable in weighing options for using stems cells in arthritis. It is very important to know the effect of aging on stems cells and how stem cell replacement might effectively treat the causes of osteoarthritis. We will establish conditions for stem cells to repair a surgical defect in laboratory models and test efficacy in animal models of cartilage defects. We will demonstrate that stem cells have anti-arthritic effects, establish optimal conditions for stem cells to migrate into the diseased tissue and initiate tissue repair, and test efficacy in animal models of arthritis. We will plan safety and efficacy studies for the preclinical phase, identify collaborators with the facilities to obtain, process, and provide cell-based therapies, and identify clinical collaborators in anticipation of clinical trials. If necessary we will also identify commercialization partners. Stem cells fight disease and repair tissues in the body. We anticipate that stem cells implanted in arthritic cartilage will treat the arthritis in addition to producing tissue to heal the defect in the cartilage. An approach that heals cartilage defects as well as treats the underlying arthritis would be very valuable. If our research is successful, this could lead to first ever treatment of osteoarthritis with or without stem cells. This treatment would have a huge impact on the large numbers of patients who suffer from arthritis as well as in reducing the economic burden created by arthritis.
Statement of Benefit to California: 
California has been at the forefront of biomedical research for more than 40 years and is internationally recognized as the biotechnology center of the world. The recent debate over the moral and the ethical issues of stem cell research has hampered the progress of scientific discoveries in this field, especially in the US. The CIRM is a unique institute that fosters ethical stem cell research in California. The CIRM also serves as an exemplary model for similar programs in other states and countries. This grant proposal falls under the mission statement of the CIRM for funding innovative research. The proposal will generate highly innovative and effective therapies for cartilage degeneration and osteoarthritis and will explore the potential use of tissue-engineered products from stem cells. If successful, this will further validate the significance of the CIRM program and will help maintain California's leading position at the cutting edge of biomedical research. Reducing the medical and economic burden of large numbers of patients who suffer from arthritis would is of significant benefit.
Progress Report: 
  • Arthritis is the result of degeneration of cartilage (the tissue lining the joints) and leads to pain and limitation of function. The annual economic impact of arthritis in the U.S. is estimated at over $120 billion, representing more than 2% of the gross domestic product. The prevalence of arthritic conditions is also expected to increase as the population increases and ages in the coming decades. Current treatment options for osteoarthritis are limited to pain reduction and joint replacement surgery.
  • Stem cells have tremendous potential for treating disease and replacing or regenerating the diseased tissue. In this grant we proposed a series of experiments to develop stems cells for use in arthritis.
  • We have met all the milestones we proposed in the first year of the grant application. We have differentiated embryonic stem cells into cells that can generate cartilage tissue similar to that generated by normal cartilage cells. We have induced pluripotency in adult human cells obtained from skin. Inducing pluripotency means transforming adult cells into cells that function very similar to embryonic stem cells. The advantage of this approach is that it removes the need for embryos as source of cells and greatly reduces the risk of rejection by the patient. We have also induced pluripotency in adult human cells obtained from joint cartilage. We believe that the original source of the cells may make a significant difference in the quality of the tissue being regenerated. For example, pluripotent cells generated from cartilage cells will likely produce a better quality of cartilage tissue than pluripotent cells generated from skin cells.
  • We have established conditions for successful repair of surgical defects using stem cells in laboratory models. We are currently working on an appropriate surgical technique for the in vivo experiments, which will involve implanting these cells in cartilage defects in live animals.
  • We have completed our experiments as outlined in our grant submission, which was the goal to enhance the development of cartilage by testing of various stem cells lines. The next phase of our project will be to prepare for the animal experiments to test the viability of our laboratory experiments that would result in cartilage repair.
  • Our initial application established the goals of our project and the reasons for our study. Arthritis is the result of degeneration of cartilage (the tissue lining the joints) and leads to pain and limitation of function. Arthritis and other rheumatic diseases are among the most common of all health conditions and are the number one cause of disability in the United States. The annual economic impact of arthritis in the U.S. is estimated at over $120 billion, representing more than 2% of the gross domestic product. The prevalence of arthritic conditions is also expected to increase as the population increases and ages in the coming decades. Current treatment options for osteoarthritis are limited to pain reduction and joint replacement surgery.
  • Stem cells have tremendous potential for treating disease and replacing or regenerating the diseased tissue. In this project our objective is to use cells derived from stems cells to treat arthritis. We have completed our experiments as per our proposed timeline and have met milestones outlined in our grant submission.
  • We have established conditions for converting stem cells into cartilage tissue cells that can repair bone and cartilage defects in laboratory models. We have identified several cell lines with the highest potential for tissue repair. We optimized culture conditions to generate the highest quality of tissue. In our initial experiments we found no evidence of cell rejection response in animals. We are now in the process of testing efficacy of the three most promising cell lines in regenerating healthy tissue in animals with cartilage defects.
  • In the next phase of our project we will plan safety and efficacy studies for the preclinical phase, identify collaborators with the facilities to obtain, process, and provide cell-based therapies, and identify clinical collaborators in anticipation of clinical trials. If necessary we will also identify commercialization partners.
  • We anticipate that stem cells implanted in arthritic cartilage will treat the arthritis in addition to producing tissue to heal the defect in the cartilage. An approach that heals cartilage defects as well as treats the underlying arthritis would be very valuable. If our research is successful, this could lead to first ever treatment of osteoarthritis with or without stem cells. This treatment would have a huge impact on the large numbers of patients who suffer from arthritis as well as in reducing the economic burden created by arthritis.
  • Our initial application established the goals of our project and the reasons for our study. Arthritis is the result of degeneration of cartilage (the tissue lining the joints) and leads to pain and limitation of function. Arthritis and other rheumatic diseases are among the most common of all health conditions and are the number one cause of disability in the United States. The annual economic impact of arthritis in the U.S. is estimated at over $120 billion, representing more than 2% of the gross domestic product. The prevalence of arthritic conditions is also expected to increase as the population increases and ages in the coming decades. Current treatment options for osteoarthritis are limited to pain reduction and joint replacement surgery.
  • Stem cells have tremendous potential for treating disease and replacing or regenerating the diseased tissue. In this project our objective is to use cells derived from stems cells to treat arthritis. We have completed our experiments as per our proposed timeline and have met milestones outlined in our grant submission.
  • We have established conditions for converting stem cells into cartilage tissue cells that can repair bone and cartilage defects in laboratory models. We have identified several cell lines with the highest potential for tissue repair. We optimized culture conditions to generate the highest quality of tissue. In our initial experiments we found no evidence of cell rejection response in animals. We are now in the process of testing efficacy of the three most promising cell lines in regenerating healthy tissue in animals with cartilage defects.
  • In the next phase of our project we will plan safety and efficacy studies for the preclinical phase, identify collaborators with the facilities to obtain, process, and provide cell-based therapies, and identify clinical collaborators in anticipation of clinical trials. If necessary we will also identify commercialization partners.
  • We anticipate that stem cells implanted in arthritic cartilage will treat the arthritis in addition to producing tissue to heal the defect in the cartilage. An approach that heals cartilage defects as well as treats the underlying arthritis would be very valuable. If our research is successful, this could lead to first ever treatment of osteoarthritis with or without stem cells. This treatment would have a huge impact on the large numbers of patients who suffer from arthritis as well as in reducing the economic burden created by arthritis.
  • Our initial application established the goals of our project and the reasons for our study. Arthritis is the result of degeneration of cartilage (the tissue lining the joints) and leads to pain and limitation of function. Arthritis and other rheumatic diseases are among the most common of all health conditions and are the number one cause of disability in the United States. The annual economic impact of arthritis in the U.S. is estimated at over $120 billion, representing more than 2% of the gross domestic product. The prevalence of arthritic conditions is also expected to increase as the population increases and ages in the coming decades. Current treatment options for osteoarthritis are limited to pain reduction and joint replacement surgery.
  • Stem cells have tremendous potential for treating disease and replacing or regenerating the diseased tissue. In this project our objective is to use cells derived from stems cells to treat arthritis. We have completed our experiments as per our proposed timeline and have met milestones outlined in our grant submission.
  • We have established conditions for converting stem cells into cartilage tissue cells that can repair bone and cartilage defects in laboratory models. We have identified several cell lines with the highest potential for tissue repair. We optimized culture conditions to generate the highest quality of tissue. In our initial experiments we found no evidence of cell rejection response in vivo. We have testing efficacy of the most promising cell lines in regenerating healthy repair tissue in cartilage defects and have selected a preclinical candidate.
  • The next step is to plan safety and efficacy studies for the preclinical phase, identify collaborators with the facilities to obtain, process, and provide cell-based therapies, and identify clinical collaborators in anticipation of clinical trials. If necessary we will also identify commercialization partners.
  • We also anticipate that stem cells implanted in arthritic cartilage will treat the arthritis in addition to producing tissue to heal the defect in the cartilage. An approach that heals cartilage defects as well as treats the underlying arthritis would be very valuable. If our research is successful, this could lead to first treatment of osteoarthritis that alters the progression of the disease. This treatment would have a huge impact on the large numbers of patients who suffer from arthritis as well as in reducing the significant economic burden created by arthritis.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00916
Investigator: 
ICOC Funds Committed: 
$2 396 871
Disease Focus: 
Bone or Cartilage Disease
Trauma
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The goal of this proposal is to develop cell-based therapies that lead to the better healing of traumatic head injuries. Our first strategy will be to use genetics and embryology in zebrafish to identify factors that can convert human embryonic stem cells into replacement skeleton for the head and face. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. As zebrafish develop rapidly and can be grown in large numbers, a growing number of researchers are using zebrafish to study how and when cells decide to make a specific type of tissue – such as muscle, neurons, and skeleton - in the vertebrate embryo. Recently, we have isolated two new zebrafish mutants that completely lack the head skeleton. By studying these mutants, we hope to identify the cellular origins and genes that make head skeletal precursors in the embryo. These genes will then be tested for their ability to drive human embryonic stem cells along a head skeletal lineage. Our second strategy will be to test whether a population of cells, similar to the one that makes the head skeleton in the embryo, exists in the adult face. We have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation. In this proposal, we use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can give rise to new head skeleton in response to injury. Traumatic injuries to the face are common, and treatment typically involves grafting skeleton from other parts of the patient to the injury site. Unfortunately, the amount of skeleton available for grafts is in short supply, and surgeries often result in facial disfigurement that causes psychological suffering for the patient for years to come. Here we propose two better treatments that would lead to more efficient healing and less scarring. The first treatment would be to differentiate human embryonic stem cells, a potentially limitless resource, into skeletal precursors that can be grafted into the head injury site. By understanding the common pathways by which head skeletal cells are specified in the zebrafish embryo and human embryonic stem cells, we will be able to generate skeletal replacement cells in large quantities in cell culture. The second treatment would be to stimulate adult stem cells already in the face to regenerate the injured head skeleton. If successful, our experiments on zebrafish jaw regeneration will allow us to devise strategies to augment the natural skeletal repair mechanisms of humans.
Statement of Benefit to California: 
Traumatic injuries to the head, such as those caused by car accidents and gunshot wounds, are commonly seen in emergency rooms throughout California. Current treatments for severe injuries of the head skeleton involve either grafting skeleton from another part of the body to the injury site or, in cases where there is not sufficient skeleton available for grafts, implanting metal plates. Although these operations save lives, they often result in facial disfigurement that causes psychological suffering for the patient for years to come. For this reason, there is enormous interest in cell-based skeletal replacement therapies that will heal the face without leaving disfiguring scars. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. Thus, we are using the zebrafish embryo to rapidly identify factors that can make head skeletal precursors, and then asking if these same factors can instruct human embryonic stem cells to form skeletal replacement cells. In addition, we have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation, and we will use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can regenerate the face. The successful completion of these experiments would allow us to both generate unlimited amounts of head skeletal precursors for facial repair and stimulate latent skeletal repair mechanisms. The combination of these approaches will lead to therapies that promote a more natural healing of the face, thus allowing Californians to eventually resume normal lives after catastrophic accidents.
Progress Report: 
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.
  • In the first year of this award, we have made a landmark discovery concerning how the skeletal-forming cells arise in the head. The head skeleton arises from an unusual cell population called the neural crest, which is characterized by its ability to form a very wide diversity of cell types in the embryo. We had previously described the isolation of two mutant lines of zebrafish that completely and specifically lack the head skeleton. We have now identified a mutation in a variant histone protein as the genetic basis for the lack of head skeleton in one of these lines. Histone proteins play a central role in not only wrapping our DNA but also controlling which genes are active in different cell types. Despite the fact that histones are expressed in every cell in the body, we have found that variant histones are uniquely required for neural crest cells to acquire skeleton-forming potential. In particular, our findings indicate that the head skeleton forms by a process of developmental reprogramming, in which precursor cells with a limited potential undergo large-scale changes in their DNA packaging such that they are able to form a much wider array of cell types.
  • Controlled cell reprogramming is becoming one of the most promising directions of regenerative medicine. For example, there is enormous therapeutic potential in being able to take cells from a patient, reprogram these cells back to a naïve state with defined factors, and then induce these cells to form replacement cells of any type that can be introduced back into the patient. Our discovery that the vertebrate embryo uses a similar process of reprogramming to generate head skeletal cells during development provides us an opportunity to better understand how reprogramming works at a molecular level. In addition, our finding that head skeletal cells form by developmental reprogramming suggests that adult patient-specific cells can be directly reprogrammed to form head skeletal precursors. Moreover, as the variant histone we have identified is identical at the protein level between zebrafish and humans, it is likely that the reprogramming process we have discovered operates in humans as well. Thus, in the coming years of this grant we are excited to develop methods of reprogramming adult patient-specific cells to a head skeletal fate, such that we can generate large quantities of replacement cells to repair the face and skull.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.
  • In the second year of this award, we have extended our initial findings that variant histones play an essential role in the generation of head skeletal cells. Skeletal cells usually arise from a cell population called the mesoderm. However, in the head a unique population of ectoderm cells, which normally forms derivatives such as neurons and skin, has an added ability to form skeletal cells. How these cranial neural crest cells acquire this extra potential remains unclear. Here we show that variant histones function within the ectoderm to give cranial neural crest cells skeletal-forming ability. In addition, we have used biochemical analysis in a human cell line to demonstrate how mutations in a particular H3.3 type of variant histone disrupt the association of histones with DNA. Furthermore, we have developed tools which allow us to analyze how variant histone changes throughout the genome endow neural crest ectoderm cells with mesoderm-like skeletal-forming potential.
  • Variant histones have been implicated in cell reprogramming, whereby a mature cell regains the ability to form many more cells that can repair a damaged tissue. As we find that variant histones are required for reprogramming of the ectoderm to form neural crest during early development, we believe that we can use this pathway to convert patient-specific cells to neural crest and skeletal cells that can be used for facial repair. To test this, we have developed a mouse model in which we can detect the ability of neural crest genes to convert mature cells to neural crest and skeletal fates. In parallel, we have developed a model of jaw regeneration in zebrafish that will allow us to test whether adult cells within the animal can be reprogrammed to repair the skeleton in response to injury. During this period, the generation of transgenic tools has allowed us to begin to address which cell types can give rise to new skeleton in response to injury. In the coming years, we hope that our work in model systems will lead to therapies for head skeletal injuries on two fronts: the generation of large amount of head skeletal precursors from patient-specific cells and the induction of increased regenerative ability of cells within the patient.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We have previously described that zebrafish with a mutant form of a variant histone H3.3 protein have very specific defects in the ability of neural crest cells to form skeleton. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In the third year of this award, we have expanded our analysis to examine how the incorporation of histone H3.3 is regulated specifically in the neural crest population. Our data suggest that such H3.3 incorporation may depend on a novel chaperone protein as reducing the function of several known H3.3 chaperone proteins does not lead to specific neural crest or head skeletal defects. A clue to what regulates H3.3 activity comes from a second zebrafish mutant – called myx - that we are studying, which has head skeletal defects similar to what we see in our H3.3 mutant. We have mapped the myx mutant interval to a very small region that contains a putative H3.3 chaperone protein. We are currently establishing whether loss-of-function of this chaperone accounts for myx defects. Together, our studies of H3.3 and myx mutants will shed light on how to generate cells with the ability to form replacement head skeleton in patients. To this aim, we have also begun experiments to use our findings in zebrafish to directly convert mammalian cells (initially mouse but then in humans) to a neural crest and skeletal fate.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that the zebrafish lower jaw bone regenerates through a cartilage intermediate, in contrast to the direct differentiation to bone during development. Hence, our findings indicate that bone regeneration in zebrafish is a cellularly distinct mechanism than bone development. Furthermore, we have found that the FGF signaling pathway is greatly upregulated during early jaw regeneration. FGF signaling also mediates the regeneration of the heart and other organs in zebrafish, and thus jaw regeneration may rely on a common regenerative program throughout the zebrafish. In the coming period, we plan to test the functional requirements of FGF signaling in mediating jaw regeneration, as well as identifying the stem cell populations that are the FGF-dependent source of new bone.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. In the previous grant cycle, we published a manuscript in PLoS Genetics describing the role of a variant histone H3.3 protein in controlling the ability of neural crest cells to form the head skeleton of zebrafish. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In addition, we published a separate study in PLoS Genetics that showed a critical role of Twist1 in guiding these neural crest cells to make head skeleton at the expense of other cell types such as neurons. Together, our studies of H3.3 and Twist1 in zebrafish will shed light on how to generate cells with the ability to form replacement head skeleton in patients. In ongoing experiments, we are using principles from our zebrafish system to directly convert mammalian cells (initially in mouse but then in humans) to a neural crest and skeletal fate.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, an unusual cartilage intermediate is able to directly make replacement bone, which is in marked contrast to the way bone is made during development. Furthermore, we have found a potentially critical role of the Ihh signaling pathway in allowing regenerating cartilage cells to directly make replacement bone. In the coming period, we plan to test the functional requirements of Ihh signaling in mediating jaw regeneration, as well as identifying the cellular source of bone-producing cartilage cells during jaw regeneration. As similar bone-producing cartilage cells may also be present in human fractures, lessons learned from zebrafish may allow us to stimulate these cells and hence augment bone repair in patients.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We had previously identified a unique role of histone replacement within the neural plate precursor cells that allows neural crest to make skeletal derivatives. In the current grant cycle, we now find that misexpression of groups of neural plate transcription factors is able to convert cells to a neural crest fate, and we are currently exploring whether this occurs by stimulating the histone replacement program we found to be so important for neural crest development. We are also testing whether similar neural plate transcription factors can convert mammalian cells to a neural crest fate, with the eventual goal to use this technique to generate an unlimited supply of patient-specific bone and cartilage replacement cells for skeletal repair.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. While we have a limited ability to repair defects in our skeleton, for example after bone fracture, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jawbone following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, cartilage cells are able to change their fate to directly make replacement bone, which is in marked contrast to the way bone is made during development. We have also identified a critical role of the Ihh signaling pathway in bone regeneration and in particular the generation of the critical cartilage intermediate. In adults lacking the Ihha protein, no cartilage forms after jawbone amputation and the jawbone fails to heal properly. Moreover, in collaborative work we find that such bone-producing cartilage cells may also be present in a mammalian model of bone healing. We are therefore excited by the prospects of using similar bone-producing cartilage cells to repair large skeletal wounds in patients.

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