Recent advances in studies of human embryonic stem cells have indicated the value of using these cells to aid regeneration of skeletal tissues. Mesenchymal stem cells that repair the skeleton suffer age-related impairments. For example, aging reduces the number, and proliferative capacity of mesenchymal stem cells which impairs fracture healing. Hence, delivering cells that have the ability to stimulate and participate in skeletal repair would greatly benefit the elderly population of orthopaedic patients. In addition, arthritis resulting from traumatic injury or aging occurs as a result of destruction of the lining of the articular surface of joints. Currently, treatment options include replacement with an artificial joint, because the cartilage lining the joints lacks an adequate intrinsic population of stem cells for regeneration. While this therapy is effective, the artificial joint is temporary and lasts 10-15 years. A better approach for treating arthritis would be to develop a living replacement joint but this relies on finding the correct cells for bioengineering the new joint. Human embryonic stem cells (hESCs) can be expanded indefinitely in vitro while maintaining their undifferentiated, pluripotent state, and these cells could potentially enhance fracture repair and provide the necessary cells for engineering a living replacement joint. However, using these cells to treat recalcitrant fractures and arthritis requires the ability to control and direct differentiation along appropriate pathways. This ability would provide a virtually limitless supply of donor tissue that has the potential for in vivo transplantation and treatment of musculoskeletal diseases. Our objective is to utilize aspects of embryonic development that are important for formation of cartilage. The majority of the skeleton is derived from an embryonic tissue named mesoderm. Hence, in the First Specific Aim of this study, we will compare mesoderm formation among a variety of different hESC lines. In the Second Specific Aim we will isolate mesenchyme from embryoid bodies and assess the extent to which a regulator of chondrocyte differentiation during development and regeneration of the skeleton. Our results will provide the basis for future studies that are aimed at maximizing and refining chondrocyte differentiation during fracture repair and joint regeneration.
Statement of Benefit to California:
Using human embryonic stem cells for therapeutic purposes requires a thorough understanding of the mechanisms that regulate differentiation of these cells. In our protocol we are proposing to examine mechanisms that underlie differentiation of human embryonic stem cells into cartilage-producing cells. Our results will form the basis for future studies that are designed to generate living artificial joints and therapeutic interventions that will aid orthopaedic patients. Ultimately, these results will benefit people by improving the quality of life of patients with degenerative or traumatic injuries to the skeleton. Developing these therapies will take considerable time, effort, and resources, and providing the means for beginning this research will further stimulate the field of tissue engineering. Due to the close proximity of our research and the biotechnology companies in California, we are in a unique position to produce potential therapies more rapidly than others. As stem cell therapies begin to reach clinical settings the need for production and testing of these approaches will provide economic stimulation an job growth within the biotechnology industry. Lastly, the impact of the success of this research will be felt personally. Skeletal injury and deterioration are painful and debilitating, and in the elderly can be life threatening. Therefore, improving the ability to heal the skeleton will likely affect every resident in California at some point during their life.
SYNOPSIS: The aim of this proposal is to direct mesenchyme generated in embryoid bodies (EBs) from hESCs towards the chondrocyte lineage by forced expression of the transcription factor Sox9, known to be important during chondrocyte specification and differentiation. The ability of such hESC derivatives to integrate in tissue in vivo will be done using chick embryo as a model, or in a fracture and osteoarthritis model in nude mice. SIGNIFICANCE AND INNOVATION: As the population ages, problems like osteoporosis and osteoarthritis become more prevalent. Developing stem cell-based or -derived therapies to repair bone and cartilage are therefore significant. The overall goal of the proposed research is to investigate the role of hESCs in generating chondrocytes that can be used clinically to repair fractures. The specific aims of the research are to (1) assess the efficiency of mesoderm differentiation of 8 UCSF-derived hESC lines in vitro and (2) investigate the role of Sox-9 in directing chondrocyte differentiation in the setting of several animal models (developmental, and traumatic) STRENGTHS: The team of investigators has significant experience in mesenchymal stem cells (MSCs), osteoprogenitor and cartilage progenitor cells, as well as with multiple models of osteogenesis and chondrogenesis; they also have extensive experience with models of bone and cartilage defects to be used and validated for their suitability to test hESC-derived bone and cartilage progenitors. The hypothesis that Sox9 will enhance chondrocyte generated from mesoderm of EBs should be correct although not particularly innovative. Aim 2 uses a variety of animal models and is the strength of the proposal in that the behavior of transplanted hESCs can be examined broadly. WEAKNESSES: The first aim is puzzling. Surely all of the lines have some mesoderm potential or they would not be offered as lines, and the data from the in-house experience should be available to the investigator. At the very least the lines have likely been injected into immunodeficient rodents to look for teratoma formation. So there must be a basis for believing they would be wildly heterogeneous in terms of mesoderm potential, but the basis for the belief is not presented. But the real puzzle is the significance of Aim 1. If the PI believes that a relatively lower expression of brachyury indicates a line that is not likely to make good or enough chondrocytes, then why not test that hypothesis? What level of difference in the percentage of cells expressing brachyury does the PI believe is significant and why? There is no provision to test the underlying hypothesis of the Aim, which reading between the lines, is that these hESC lines have significant inherent differences in their potential for mesoderm formation. Aim 2 uses a variety of animal models and is the strength of the proposal in that the behavior of transplanted hESCs can be examined broadly. There does not, however, seem to be a provision for purifying the Sox-9 expressing cells for use in these models. Since the cells have eGFP, could they not be sorted by FACS and expanded, making the analyses cleaner? Otherwise a mixed population of cells is being injected into the animal models. The exact end-points of analysis in the animal studies are not well presented. It would be nice to know how the investigators will analyze for cells that migrate away from where they are placed. In particular in the migratory stream of the chick, the cells could end up in a lot of places with a lot of different fates, and that would tell us a lot about the behavior of hESCs under potent developmental cues. In the fracture studies, there is no mention made of looking at integrity of repairs except histologically. Given the huge orthopedic research experience in this group of collaborators, the models are not used to their fullest, especially given their translational potential. It is unclear why the investigator believes that a mesodermal tumor will form only in the thigh of the rodent fracture model, but not in any other of the models. Does varying the expression of Sox-9 (using pure lines of expressing cells vs. mixed lines) make a difference in the tumor formation? That would be a potentially informative question faciliatated by the reagents developed for this proposal. A number of other concerns were raised. Difficulties with stable overexpression from lentiviral vectors are not addressed. As studies will not compare classical precursors for bone and cartliage, namely MSCs, the investigators will not be able to conclude that hESCs are a better source of cells for these defects than adult cells. Finally, no methods are provided on how the investigators plan to purify cartilage and bone precursors and how they will avoid teratoma formation. DISCUSSION: There was no discussion following the reviewers' comments.