Development and Evaluation of Human Stem Cell-Derived Neurons for High-Throughput, Automated Electrophysiology.
Tools and Technologies I
Human stem cell-derived neurons (hSCNs) represent powerful, physiologically relevant cells for drug discovery, yet there is limited data on the proteins that form ion channels. Ion channels are the site of action of many therapeutic drugs and highly attractive targets for the development of new medicines for an array of disorders including epilepsy, dementia and stroke. Ion channels also represent under-exploited drug targets primarily because their investigation depends on skilled electrophysiologists conducting time-consuming experiments with low data throughput for drug discovery. These technical challenges represent major bottlenecks to the identification and development of novel agents for neuropsychiatric disorders. Characterization of the ion channels present in hSCNs combined with the development of tools for high-throughput assays would therefore be a major advance in using stem cells in the drug discovery process. Recently, several automated recording systems have been developed which greatly increase the number, speed and array of ion channels that can be studied. However, these automated machines are limited to the use of experimental cells expressing cloned channels. Recent data show that hSCNs express protein markers and ion channels found in native neurons and these nerve cells can be grown and maintained in culture over long periods rendering them amenable to automated high speed investigation: development of hSCNs for automated physiological studies would thus enable simultaneous study of multiple ion channel drug targets in a single (standardized, human) neuron and greatly increase the sophistication of neurophysiological and neuropharmacological experimentation. This proposal therefore aims to develop the methods and evaluate the suitability of human stem cell-derived neurons for automated patch-clamp electrophysiology studies. The integration of stem cell technology with automated systems would be unique in California and yield significant improvements in ion channel screening, lead optimization and assessment of drug safety; it will also later provide key information on the consequences of genetic mutations in ion channels and the impact of culture conditions to guide the generation of specific cell type derivatives. The combination of automated electrophysiology with hSCNs for ion channel studies will significantly enhance the value of stem cells as tools in the drug discovery and biotech industry in California.
Statement of Benefit to California:
This proposal aims to develop the methods and evaluate the suitability of exploiting human stem cell-derived neurons for automated, high speed physiological studies of ion channels for drug discovery. Ion channels in nerve cells are highly attractive but under-exploited targets for the development of new medicines because they cannot be screened easily. Success in this project will optimize the identification of new drug leads for neuropsychiatric illnesses and improve assessment of drug safety. These are major benefits to the citizens of California. The biotech and pharmaceutical industry are significant contributors to the State economy and major employers in California. Leading the exploitation and development of stem cells as tools for drug discovery and development here will support job creation and significantly benefit our economy. Utilization of nerve cells derived from human stem cells will also lead to a significant reduction in the need for experimental animals; this is also of great benefit to the people of California.
This team of investigators proposes to develop automated patch clamp electrophysiology to study ion channels in human stem-cell derived neurons (hSCNs), with the aim of identifying and developing novel agents for psychiatric disorders. They propose two aims, largely focusing on optimal culture age and cell density conditions for the automated patch clamp work, and the identification of major voltage and ligand gated ion channels in these cells. Success in this research would not be broadly enabling, although reviewers agreed that it could lead to an enhanced ability to screen for drug candidates and assess drug safety in neurological disorders. Reviewers noted that the PI has already established that hSCNs derived form this particular cell line develop many of the molecular and physiological properties of neurons. In addition, the PI has shown that these hSCNS express voltage gated ion channels. It appears that this research would confirm these earlier findings, and extend them to enable high throughput testing and evaluation of the cells’ physiological properties. The investigators suggest that the project will allow the use of human stem cell derived neurons for analyzing the consequences of channelopathies at high efficiency. They argue that this would be of value because there is limited information available currently on ion channels in such cells, and because use of human cells would reduce the use of animals. Reviewers felt that these goals are important but modest, and the research was not particularly novel or innovative. The application consists of a straightforward research plan that has two highly technical specific aims. Reviewers commented that the rationale for the proposed research is logical and convincing and the research plan is clear and designed appropriately, but the endpoint and milestones of the project were unclear. In addition, there are a number of limitations of the technique that are neither acknowledged nor addressed in the application. First, although it is stated that the planar patch clamp will be compared to conventional patch clamp, the parameters that will be compared or what action will be taken if the comparisons yield large divergence are not discussed. The applicant does not discuss what approaches will be taken if it is difficult to obtain gigaohm seals with high frequency, or if cells become unhealthy during manipulation. Second, reviewers raised questions about the neurospheres that will be studied - the applicant describes how hSCNS will be grown both as neurospheres and monolayers, and cellular samples from both types of cultures will be tested. Certainly, the neurospheres these investigators have developed might be useful, but there is no discussion about heterogeneity of cell types in these spheres. Furthermore, it is not entirely clear what the advantage of dissociating cells from neurospheres might be, as compared to conventional dissociation from a monolayer. Third, the drugs that will be tested and the channels that will be examined are not detailed. Fourth, the study focuses entirely on electrophysiological approaches - molecular techniques that could provide another level of information and support electrophysiological measurements are not employed. Finally, although the system may increase throughput, there are only 16 channels - this is a long way from high-throughput, according to one reviewer. Although this system would be useful, its utility depends on the long-range goals of the investigation that, as discussed above, were considered modest. Overall, the research plan was technically feasible and appropriately designed, although it lacked conceptual details and the applicant did not adequately address the limitations of the electrophysiological technique to be used. In conclusion, reviewers commented that this was a straightforward and feasible project that would have a very narrow impact. The team was considered adequate but not highly productive, and the research proposal lacked an endpoint, milestones and a detailed outcome plan, and did not consider likely obstacles to success.