Tools and Technologies II
$1 803 325
A current roadblock for human stem cell therapies is the lack of efficient cell sorting methodologies to produce purified cells for transplantation. Stem cell transplants should contain purified cells designed to provide functional recovery but lack cells that can form tumors, making pure cell populations a necessity for the stem cell field. Conventional techniques rely on the presence of specific cell surface markers, which have not been identified for many stem cells of potential therapeutic interest. We have addressed this problem by developing a new sorting method using microfluidics and a technology termed “dielectrophoresis” (DEP) that utilizes electric fields to separate cells. Our data show that DEP distinguishes neural stem/progenitor cells (NSPCs) from further differentiated cells, can be used to enrich NSPCs biased to make neurons, and has no deleterious effects on human NSPCs. DEP is likely to be an optimal method for sorting stem cells prior to transplantation for several reasons. DEP-based cell sorting requires no labeling, so cells isolated in this manner will not be coated by antibodies immediately prior to transplantation, potentially reducing immune-mediated rejection of sorted cells. Furthermore, cells can be sorted by DEP through multiple channels with distinct DEP force profiles to refine separation in a single sort; a similar strategy using other methods would require additional time to label the cells with antibodies against a different set of markers. Our data show that DEP does not alter or harm human NSPCs and the recovery and survival of these cells after DEP is superior to that of cells isolated by other methods. Importantly, initial studies show that isolation of NSPCs by DEP generates greater enrichment of certain cell types than other methods. Current sorting methodologies using cell surface markers are insufficient for purifying many stem cell populations, including neuron-restricted progenitors in the neural lineage, due to a lack of necessary specificity of available markers. The goal of our proposal is to improve the sorting of stem cells prior to transplantation by developing a novel and label-free method complementary to currently used marker-based techniques.
Statement of Benefit to California:
The goal of this project is to determine whether a novel strategy using dielectrophoresis (DEP) can serve as a complementary and alternative approach to marker-based separation of stem cell subpopulations to be used for transplantation. In the course of these studies, we expect to remove tumor-promoting cells from neural human progenitor cells to improve their safety for transplantation. We also hope to make purified populations of cells that can replace specific central nervous system cells. We hope this label-free method for isolating stem cell subpopulations will greatly increase the speed of stem cell therapeutics in California.
This proposal aims to further develop a novel high throughput sorting technology that is based on a platform utilizing microfluidics and dielectrophoresis (DEP), which utilizes electrical fields to separate cells. The specific focus of this application is to use the technology to sort neural stem/progenitor cells (NSPCs) and subsequently, to test their efficacy in a transplantation model. The technology will be benchmarked against the current state of the art sorting technology. The Principal Investigator (PI) proposes two Aims: 1) to develop DEP separation devices that achieve high throughput and cell purity; 2) to determine if DEP sorting enhances the homogeneity of cellular phenotypes in transplants. Reviewers agreed that the application addresses a critical translational bottleneck. There is a clear need to improve sorting methods to separate cells from a heterogeneous population, both to remove mis-differentiated cells and purify the functional ones. Reviewers also found the proposed platform combining microfluidics and DEP to be innovative, especially the design of the proposed devices and the novel sorting approach for increasing throughput without increasing shear. The proposed DEP sorting approach is label-free which is a potential significant advantage of the technology. The reviewers liked the approach of iterative technology development/improvement and functional testing. They were enthusiastic that the PI plans a direct comparison of DEP sorting against flow cytometry and praised the comprehensive functional studies. However, they expressed some reservations about feasibility given that there was no compelling preliminary data that conclusively shows useful sorting despite several years of research in the field, including research funded by CIRM. Specifically, reviewers noted that much of the preliminary data describes correlations between electrical properties and different cell types, but commented that the utility of the approach must come in sorting, not just analysis. There was very little compelling preliminary data presented on sorting of a mixed population of cells demonstrating useful enrichment or subtype separation raising questions about the actual performance and efficiency of the system. A reviewer expressed the concern that the lack of preliminary data establishing the basis (e.g. cell size) for DEP-based cell isolation raises the possibility that the proposed research will not result in a useful technique. In addition, although reviewers agreed that the proposed innovative strategies would increase throughput, there was some concern that the applicant had not addressed the issue of cell aggregation at high cell concentrations and its impact on purity and recovery. They noted that, in general, the applicant did not discuss the required yield/recovery of the separation technology, which is as important as purity and throughput. One reviewer urged the applicant to carefully further consider their validation studies citing concerns that the proposed marker for neural stem cells was too widely expressed on proliferating cells which could confuse results and that the NSPC differentiation efficiency would not result in sufficient cells to conduct transplantation studies. Reviewers noted that the PI is very well respected and a prolific researcher with publications in both the stem cell and microfluidics fields. They praised his/her willingness to commit 50% effort to the project and the strength and relevant expertise of the research team. They judged the environment to be excellent. In summary, this application describes the development of a novel platform that combines microfluidics and DEP to sort NSPCs. Although the application addresses a key translational bottleneck and proposes novel and innovative technology, reviewers’ enthusiasm was diminished based on concerns about feasibility of the technology and the research plan.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.