Long-term Organotypic Slice Model: A novel ex vivo culture system for the in situ study of stem cell and cancer cell biology in the brain and spinal cord
Tools and Technologies I
Novel applications for Stem Cell Therapy have been proposed for a broad range of congenital and acquired pathology. The Central Nervous System represents a key target for stem cell therapy. The central nervous system, comprised of the brain and spinal cord, has long been viewed as neither capable of clinically significant regeneration nor, unlike many other vital organs, amenable to reconstitution through transplantation. Stem cell therapy may provide the first meaningful approach for the reestablishment of neurological function in the setting of inherited or developmental absence, or in meaningful recovery after loss of function for addressing brain and spinal cord pathology. Malignant brain and spinal cord tumors remain a leading cause of seriousness and death 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. 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. New approaches to the treatment of brain tumors are desperately needed. New insight into the nature of cancer has resulted from the isolation and preliminary characterization of cancer stem cells from a number of malignancies including leukemia, multiple myeloma, squamous cell cancer, malignant melanoma, breast cancer, and in the brain: medulloblastoma, ependymoma and malignant glioma. The nature of their origins from their normal stem cell counterparts will provide invaluable new insights into the development of cancer, leading to novel treatment strategies. This proposal focuses on a new way of studying stem cells, including cancer stem cells that may lead to new insights into the development of brain and spinal cord cancers, leading to improved treatment.
Statement of Benefit to California:
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. 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.
The primary goal of this project is to develop an in vitro model system using brain tumor slices to study stem cells derived from pediatric brain and spinal cord tumor patients. The applicants will generate induced pluripotent stem (iPS) cells from skin fibroblasts of patients with brain tumors, as well as normal controls, for culture and analysis in the tumor organotypic slice model. The applicants will also screen a chemical library in a rodent organotypic brain slice for effects on iPS cell interactions with tumors for neural fate determination. Reviewers found this an unfocused proposal that does not establish a rationale, a hypothesis, or research plan to overcome a significant roadblock. The aims are disconnected and do not appear well suited to solve either a clearly defined problem in stem cell biology or a clinically relevant question in cancer biology. There is no attempt to clearly identify brain cancer stem cells, nor is it clear how the general interaction of other stem cells (embryonic, iPS, endogenous neural stem cells, or mesenchymal stem cells) with the brain tumor slices will actually be identified or analyzed in an informative manner. The isolation of iPS cells from skin cells of brain tumor patients in the long run could prove a valuable resource, if it were the case that individuals had significant genetic predisposition to develop malignancy. Reviewers felt that no evidence was presented that this is the case for the patient group in question, nor was a plan presented for genotyping. The group is far too small to be useful for this purpose unless there were familial cases of the disease. Collections of fibroblasts that could be subsequently utilized for generation of iPS cells, if genetic predisposition were in fact to be demonstrated, would be a much less costly approach to achieve the same goal of archiving cells, pending a compelling reason to generate the iPS lines. The applicant proposes, reasonably enough, to direct differentiation of such iPS cells towards a neural fate. However, reviewers felt that there was no clear rationale or plan to assess how the cells from cancer patients and those from unaffected controls might differ. The high probability that somatic mutations are necessary for brain tumorigenesis was not considered – if this is the case, the generation of iPS cells would not capture the tumor cell genotypes. Moreover, reviewers noted that the introduction of vectors carrying oncogenes (required to generate iPS cells) is likely to prove a major confounding variable in the analysis of intrinsic tumorigenicity of the cells with the patient's genotype. This essentially obviates the value of the collection of the patient specific iPS lines. It might be possible to use known markers of neural stem cells and of putative brain tumor stem cells, such as CD133, to isolate patient-specific cancer stem cells that could be compared with the neurally-driven iPS cells. This type of approach was not considered. Reviewers viewed the identification of small molecules that might induce neural lineage differentiation of human embryonic stem cells (hESC) or iPS cells as a worthwhile goal that would advance stem cell biology. However, reviewers felt that the proposed approach of screening a chemical library using hESC and iPS cells on the organotypic brain slices did not appear to be a logical way to achieve this goal. Reviewers considered the principal investigator (PI) a well-qualified physician with expertise in brain neoplasms. The PI’s research track record does not indicate corresponding expertise in stem cell biology or fundamental cancer cell biology. Key personnel appear generally competent in molecular and cell biology, but lack deep expertise in specific areas of study relevant to the proposal. Collaborators include excellent individuals, but these do not rescue the team or the poorly developed research proposal. Overall, reviewers commented that the combination of an interesting culture model (the organotypic brain slices) with "trendy" tools (iPS cell generation and chemical libraries) will have no meaningful impact without a much clearer rationale and plan.