Tools and Technologies I
Stem cells are typified by their ability to differentiate into a wide range of specific cell types, including nerve cells, brain cells, and other cells that do not typically regenerate in the human body. This pluripotency holds great promise for regenerative medicine, possibly enabling the repair of damaged or defective tissues in adults and children. A great promise for stem cells is the production of very large quantities of stem cells differentiated into new heart muscle cells, skin cells, nerve cells, or other types of cells that are needed to repair these tissues. Controlling what type of cells stem cells differentiate into, and separating those that have differentiated into distinct types, from those that have not differentiated, is a key element in using stem cells as therapeutic elements. Control is typically explored using a wide range of different chemical and physical environments, and is proving a highly complex and challenging area of research. Distinguishing and purifying the results of these control experiments, both in research and in possible clinical application, will clearly be a significant and important part of the eventual therapeutic use of stem cells. The technologies we propose to develop would enable the very high throughput and highly specific separation of differentiated and non-differentiated stem cells into separate groups, so that very highly purified samples of any particular type would then be available for further development or for direct therapeutic use. The instruments are based on technology that would allow very inexpensive, disposable, yet highly sensitive stem cell identification and purification, making the instruments readily and cheaply available to both the researcher and the clinician. It is based on very novel and disruptive technology, quite distinct from the methods presently used for these applications, providing a route to much simpler and faster instruments, and more rapid results, than present methodologies.
Statement of Benefit to California:
We will develop a high-throughput human stem cell sorter, using a combination of microfluidics, biofunctionalization, and radiofrequency velocimetry. The resulting device will provide a label-free method to sort billions of stem cells per hour, based on their surface protein expression. The end product will be a small, hand-held unit that includes disposable components, whose operation will be simple, with software-definable discriminants driving multi-axis sorting decisions. Such a device will have immediate applications to the identification and purification of heterogeneous stem cell populations, and to the identification and removal of cancer stem cells. The benefits to California of this research are multi-dimensional. To begin, the technology produced through this research will have a direct impact on stem cell research and use, an area to which California has clearly made a strong commitment; commercial and academic research efforts within California would benefit directly from this tool. Second, the technology produced here will likely form the basis of a commercial enterprise to produce and further develop this technology. This enterprise will most likely be based in California, and if successful would provide employment, new technology, and tax revenue to the state. Third, the participation of the two PIs, bringing highly distinct expertise in biophysics (Cleland), and cellular biology (Kosik), will provide a unique combination of backgrounds and technologies that will likely generate new ideas and more applications for this type of instrumentation. Fourth, the research efforts of the two postdoctoral scientists and a graduate student will train three new professionals in this cross-disciplinary area, students that will doubtless continue to work on closely related areas of work and further develop the concepts and technology that will enable future progress. Finally, this will further the development and unification of microfluidic technology with electronics, an area of technology that will likely expand further over the next decade.
This proposal is focused on the development of a novel cell sorting device that could separate stem cells by phenotype without the need for molecular labeling. The device consists of a microfluidic channel that has been functionalized to interact with specific cell-surface molecules. Cells with different combinations of surface features will interact differentially traveling through the channel and emerge at different rates. In addition to a single channel device, the applicants will develop a multi-channel version for more advanced sorting capabilities. Finally, the applicants propose to test the new device by sorting stem cells and performing phenotypic analysis. The reviewers appreciated the conceptual innovation of this proposal but felt that the capabilities of the proposed device were oversold. In addition, the proposed validation studies were inadequately described. Finally, the reviewers felt that the applicant failed to properly motivate the need for this technology in comparison to competing instrumentation. The impact of the proposed device was questioned by the reviewers, who felt that several of its capabilities were significantly overstated. In theory, such technology would be enormously useful for obtaining large quantities of pure stem cell populations, a key roadblock in the field. The ability to eliminate use of cellular labeling and minimize the impact of spurious biological interactions is an additional key advantage of the approach. However, the reviewers felt that the applicant did not sufficiently present a convincing case for the superiority of the proposed device over competing, alternative technologies. Reviewers doubted the feasibility of this proposal due to several major weaknesses in the research plan. In terms of the device construction, concerns were raised that the applicant underestimated the challenge of some features of device fabrication and optimization of component performance. The applicant also did not provide sufficient details for reviewers to understand the proposed biological validation studies. Although candidate cell surface antigens were mentioned, there was no discussion of which would be chosen for investigation, or why particular markers would be used in the validation. There were no experiments to verify that cells sorted by the new methodology are comparable to those sorted by immuno-phenotyping methods or to indicate how the sorted cells would compare with undifferentiated human embryonic stem cells (hESC). Furthermore, the reviewers felt that the applicant underestimated the scope and complexity of the proposed phenotypic analyses, and a large database would likely be required to make any sense of their findings. The reviewers felt that the research team was well qualified to pursue the proposed research but were concerned with apparent lack of insight as to how the team’s unique set of skills would be utilized appropriately to improve the current state of the art. The principal investigator is a leading physicist who has collaborated with biologists previously, and the co-investigator has miRNA expertise and has worked with stem cells. The reviewers felt that the proposal would benefit from better coordination between these collaborators to design a more detailed plan for the proposed phenotypic analyses. The budget was judged to be appropriate, although one reviewer was concerned about a possible error. The requested sum for equipment seemed extremely low, and it is possible that the applicant accidently switched this figure with that for the materials and supplies. In summary, this was an interesting proposal for a conceptually intriguing technology, but the reviewers were unconvinced of its feasibility, and therefore, its potential impact.