A universal ex vivo/in vivo hybrid platform for the in situ tracking of behavior and fate determination of defined stem cell populations: A bridge towards clinical application of stem cell therapy for CNS pathology
Early Translational I
Novel applications for Stem Cell Therapy have been proposed for a broad range of congenital and acquired pathology. The Central Nervous System (CNS) represents a key target for stem cell therapy. Malignant brain and spinal cord tumors remain a leading cause of morbidity and mortality for children and adults. Pediatric brain tumors are second only to leukemia as the most common malignancy of childhood and now represent the leading cause of cancer-related death in children. Accumulating data documents permanent functional disability exhibited by the few fortunate survivors. Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig’s disease, currently affecting Stephen Hawkins) is a progressive and usually fatal, neurodegenerative disease ultimately resulting from the loss of motor neurons. This leads to progressive muscle weakness and atrophy throughout the body. ALS is one of the most common neuromuscular diseases worldwide, affecting people of all races and ethnic backgrounds, with an incidence of approximately 2 per 100,000 annually. New approaches to the treatment of brain tumors and ALS are desperately needed. A universal platform is needed for realistic, rapid and cost-effective pre-clinical development and testing for human stem cell therapies aimed at specific diseases, such as brain tumors and ALS. The current dilemma of stem cell research arises from attempts to extrapolate results from two disparate techniques. Classically, stem cell development and survival has been solely characterized by describing the fate of dissociated cells grown on highly artificial plastic tissue culture plates. The artifactual nature of this completely foreign tissue culture system with the resultant conflicting results is becoming well recognized, as evidenced by confusing and contradictory results reported by different researchers. Pre-clinical testing of stem cell therapies has classically been evaluated by transplanting stem cell populations into animal disease models to observe for clinical improvement. This essentially represents a process in which stem cells are introduced into a “black box” with the hope of observing some desirable outcome exhibited by the transplanted animal. Here, we propose a universal hybrid platform, which allows for the tracking of stem cell fate after transplantation of these stem cell populations into small slices of brain or spinal cord, replicating human brain tumors and ALS, respectively. Finally, the fate of the patient’s own stem cells will be characterized when reintroduced into their own brain tumor containing brain slices. Through the application of the systematic stem cell investigation paradigm proposed in this application, it is hoped that more reliable pre-clinical assessment of stem cell fate and subsequent biological outcome will translate into improved predictability enabling more rapid development of efficacious, disease-specific stem cell clinical therapies.
Statement of Benefit to California:
Malignant brain and spinal cord tumors remain a leading cause of morbidity and mortality for children and adults including California. Pediatric brain tumors are second only to leukemia as the most common malignancy of childhood and now represent the leading cause of cancer-related death in children. The prognosis for malignant brain tumors remains dismal, best appreciated in poor long-term survival statistics. Accumulating data document permanent functional disability exhibited by the fortunate survivors. The costs for the patient and family cannot be overestimated. Overall estimates of the incidence of brain cancers in the United States show that about 20,000 will be diagnosed annually with about 2500 in California. The economic costs are high. Repeated use of physician, inpatient, outpatient and laboratory services as well as lost future earnings and occurrence of secondary diseases cost Californians of more than 1.5 billion dollars annually. Fundamentally new approaches to the treatment of brain tumors are desperately needed. The objectives of this proposal focus upon utilizing a refined biological model to allow for the direct study of in situ behaviors of stem cell (neural stem cells, embryonic stem cells, induced pluripotent stem cells) and cancer stem cell populations within brain and spinal cord microenvironments with the ultimate goal being improved therapeutic applications.
This is a proposal to determine whether organotypic slice models from brain and spinal cord can be used to predict the fate of human embryonic (hESC) or adult stem cells introduced into human patients, as the applicants feel the slice model is an important preclinical, predictive assay tool. In the first aim, stem cells will be injected into organotypic slices derived from human tumor specimens from the central nervous system, or from spinal cord sections from a mouse genetically altered to model amyotrophic lateral sclerosis (ALS). Migration, differentiation and survival of the cells will be tracked using confocal and multiphoton microscopy (MPM) and optical coherence tomography (OCT). In the second aim, a fiber-based multiphoton microscopy (MPM) system with a miniature probe will be used to perform in vivo imaging and tracking of stem cell in animal models. Reviewers agreed that the disease targets chosen (ALS and pediatric tumors) are important, but felt that the applicants had not made a case that the research planned would positively impact the progress toward cell therapies for these (or other) disorders. The applicant did not discuss a compelling rationale for stem cell therapies for pediatric cancer (though they may be important), nor for the use of tumor material as a good model for cell transplantation studies. Reviewers felt they had to read between the lines. In the case of ALS, reviewers understood the rationale for the slice model, as stem cells may be a good candidate for repair and restoration of motor neurons that are lost in this disease. The behavior of human embryonic stem cells (hESCs) cultured in vitro likely does not mimic their activity in vivo, where tissue-specific environmental factors and stromal elements modify their phenotype. So in principle, reviewers agreed with the applicant that comparison of the properties of hESCs in their slice model with the behavior after injection into the brains or spinal cords of mice might inform one of the utility and predictive powers of the slice model, noting that organotypic slide models from rodents have been used for a long time, but the comparisons (sufficient preliminary data) needed to justify the proposal were not presented clearly. Reviewers commented that it was difficult to assess the feasibility of the project as the proposal lacked clarity and was extremely disorganized. The applicant often referred reviewers to published data rather than providing a brief description of the intended experiments, stem cells and assays to be used, and follow-up experiments to be performed, and much of the research design and methods seemed to be derivative. Preliminary data were lacking (a major issue since the comparison data between slice and in vivo transplants are the core of data needed to convince reviewers of the necessity of developing a new preclinical model). For the first aim, the attributes of the organotypic slice cultures are described but no relevant data are provided. The second aim places a great deal of emphasis upon developing an imager, but again preliminary data are not presented and if such an instrument is not forthcoming the entire aim will be difficult to execute. Reviewers commended the principal applicant’s academic credentials and training, and commented that the listed collaborators are well-qualified and the research environment is excellent. However, they also noted that the application did not highlight the organotypic slide experience of the investigators, which contributed to concerns about the project’s feasibility. In summary, this proposal attempts to address the bottleneck of understanding behavior of cells after transplantation, devising a model to predict reliably stem cell behavior, using ALS and pediatric tumor models as test-beds for the organotypic slice work. The application did not highlight its assets in a way that reviewers could be convinced that the project was worth investment, and reviewers pointed out many weaknesses, including a diffuse and vague focus, lack of important details and preliminary data. Without the relevant preliminary data and absence of a discussion of significant experience with organotypic slice model when the roles of personnel were discussed, the reviewers had major concerns about feasibility of the project.