Pluripotent stem cells have two remarkable properties: they can divide seemingly endlessly and therefore produce large quantities of identical daughter cells, and they can also be induced to mature into virtually any cell type that makes up the human body. They are thus very useful as a source of material for biomedical research and potential medical applications. However, until recently, these cells have been hard to come by. Now, through a relatively simple process, pluripotent stem cells can be produced from virtually any cell type, including skin cells. Because collecting skin cells does not cause a person any harm we can use these to make a wide variety of cell types from individuals with specific diseases. Using this strategy it is therefore possible to take skin cells from an individual with an inherited brain disorder, turn these into brain cells, and use these to study the causes of the patient’s disease in the laboratory without ever having to touch the patient’s brain. The enthusiasm that this remarkable achievement generates, however, is tempered by the fact that finding the right environment to support development of fully functional brain cells grown in a dish in the laboratory has been difficult. In this study we will use the fact that recent studies have shown that pluripotent stem when injected into the brains of very young mice can integrate into host brain tissue and form functional brain cells. Thus we will use the animal’s brain to do what we cannot now do in the lab, make fully functional brain cells from the pluripotent stem cells. By using cells taken from patients with a specific inherited disease of the brain, therefore, we will be able to study proper brain cells and their disease in their natural environment, the brain, and, by doing so, develop new treatments for that disease. For our initial studies, we will begin by looking at the disease Fragile X syndrome, because of its unique properties and because it is the single leading cause of autism.
Statement of Benefit to California:
The autism spectrum disorders represent the single leading cause of intellectual disability (ID) in children. National annual costs exceed $35 billion, a figure that is expected to rise to $400 billion in the next decade. Although the evidence for a genetic component of ASDs is strong, identification of the gene or genes responsible has been difficult, suggesting that many different genes may play a role. Indeed, several genes have already been identified but most of them, individually, account for only a very small proportion of cases of ASD. Fragile X syndrome, on the other hand, not only represents an identified gene defect that is responsible for the most cases of intellectual disability in boys, about 40% of boys with FXS also have ASD, suggesting a very strong connection between the two IDs. Our studies intend to capitalize on this connection by developing a research method, based on analysis of neurons derived from induced pluripotent stem cells, which can be used to define the mechanisms underlying FXS and that contribute to ASD in this class of patients. If successful, we will be able to extend these studies to other causes of ASDs and provide, based on solid science, new therapeutic targets for this enigmatic class of childhood developmental disorders.
The applicants aim to create humanized animal models of complex neurodevelopmental disorders to enable study of the underlying cellular defects. Fragile X Syndrome, a neurodevelopmental disease associated with autism and caused by CGG-repeat expansion of the Fragile X Mental Retardation gene 1 (FMR1), will be used to establish the system. The applicants plan to use differential fluorescent tagging of patient fibroblasts to distinguish induced pluripotent stem cells (iPSC) bearing different mutant forms of FMR1 from individuals mosaic for expansion length in this gene. They will then transplant these iPSC into neonatal mouse brains and assess the impact of these mutations on the anatomic and physiological properties of human neural cells differentiating in vivo.
Significance and Innovation:
- Reviewers generally felt that this proposal is innovative, with a potential to start addressing the mechanisms underlying complex conditions such as autism that have been quite difficult to study.
- The rationale behind the plan to transplant undifferentiated iPSC into immunodeficient mice was deemed weak owing to the documented propensity of these cells to form teratomas in this context.
- One reviewer voiced concern regarding the ability of the mouse to recapitulate human circuit assembly and therefore questioned the relevance of this work to human pathophysiology.
Feasibility and Experimental Design:
- Reviewers appreciated the idea to differentially mark iPSC lines from a single patient mosaic for FMR1 mutations to enable their simultaneous study in the same brain. However, they highlighted serious feasibility issues with the selected approach.
- The panel was unconvinced the poorly described vectors for differential labeling of patient fibroblasts would not be silenced by the reprogramming process. Preliminary data supporting labeling utilize human embryonic stem cells, rather than hiPSC, and therefore do not allay this critical concern.
- A reviewer noted that typical reprogramming-induced genetic variability amongst iPSC lines from the same patient could confound the identification FMR1 mutation-dependent phenotypes. The applicants should describe a more robust iPSC quality control scorecard to prevent this problem.
- Variable engraftment efficiency between cell lines could complicate interpretation of the results.
- Reviewers agreed that solid preliminary data has been provided to support the remaining elements that would be required to carry out the proposed experiments.
Principal Investigator (PI) and Research Team:
- Reviewers agreed that together, the PI and co-investigator have the appropriate expertise to undertake this work and demonstrate the required level of commitment.
- A discussant noted the bulk of the work appeared planned for the co-investigator’s laboratory rather than the PI’s.
- There were some reservations that the two groups are at the early stages of collaboration on this project.
Responsiveness to the RFA:
- Reviewers agreed that the proposed studies employing human cell reprogramming to elucidate mechanisms of Fragile X syndrome disease are fully responsive to the RFA.