Novel Separation of Stem Cell Subpopulations

The main goal of our project is to identify a novel method to accurately separate stem cells and their differentiated progeny without the use of cell type-specific markers. The sorting method we are investigating uses dielectrophoresis (DEP), which generates movement of cells toward or away from electrode arrays via non-toxic electric fields. The frequency of the field and the inherent properties of the individual cells provide the basis for separation of unique cell types. Our previous data showed that DEP was capable of distinguishing mouse neural stem/precursor cells (NSPCs) from more differentiated neural cells (neurons and astrocytes). Furthermore, the dielectric properties of NSPCs that preferentially formed neurons were distinct from those of their glial-biased counterparts, which is important since these NSPC subpopulations cannot currently be discriminated by markers. The specific aims of our project are to determine the applicability of DEP-based separations for neural stem cell populations by generating novel DEP sorting devices and using them to isolate NSPCs on the basis of their fate potential. During the course of the first year on the project, we have designed, fabricated, refined, and tested a novel DEP separation device that utilizes perpendicular microfluidic channels controlled by a set of individual valves. This device is now routinely used in our NSPC separation experiments. We have also modeled and designed a continuous sorting microfluidic DEP device to provide high throughput separation of cells. We are currently at the end stages of fabrication of this device and will continue its testing and refinement in the second year of the project. Separation of NSPCs for both research and therapeutic applications requires that the cells are not altered by the isolation procedure. We have systematically and carefully tested whether the forces used for DEP-based sorting would have any adverse effects on the cells and found that DEP is actually much gentler than current sorting technologies, demonstrating the safety of DEP for NSPC separation. We have used two different DEP sorting devices to isolate subpopulations of NSPCs by DEP frequency response and in both cases found that neurogenic NSPCs are enriched by high frequency DEP-based separation. We have expanded our studies to NSPCs derived from human ES cells and discovered that the dielectric properties of these cells also reflects their fate bias. Experiments in the second year of the project will test the capacity of DEP for sorting of these cells. Successful isolation of human NSPCs that specifically generate neurons by our non-toxic and label free procedure will provide purified populations of these cells for basic biological studies, therapeutic approaches, and as a source of human neurons for drug testing.

The main goal of our project is to identify a novel method to accurately separate stem cells and their differentiated progeny without the use of cell type-specific markers. The sorting method we are investigating uses dielectrophoresis (DEP), which generates movement of cells toward or away from electrode arrays via non-toxic electric fields. The frequency of the field and the inherent properties of the individual cells provide the basis for separation of unique cell types. Our previous data showed that DEP was capable of distinguishing mouse neural stem/progenitor cells (NSPCs) from more differentiated neural cells (neurons and astrocytes). Furthermore, the dielectric properties of NSPCs that preferentially formed neurons were distinct from those of their glial-biased counterparts, which is important since these NSPC subpopulations cannot currently be discriminated by markers. The specific aims of our project are to determine the applicability of DEP-based separations for neural stem cell populations by generating novel DEP sorting devices and using them to isolate NSPCs on the basis of their fate potential. During the second year on the project, we finalized production of a multi-channel device with perpendicular channels controlled by valves. This device proved to be quite robust and was used to sort specific cell populations from NSPCs without the use of markers. To determine the applicability of DEP-based separation for stem cells, we have tested whether exposure to DEP alters the cells, sorted cells by DEP frequency and tested their purity, and applied DEP to human NSPCs. Our experiments have shown that DEP exposure does not harm NSPCs when shorter exposures as needed for cell sorting are used. At longer exposure times, particular frequencies damage ~30% of the cells. Importantly, DEP exposure does not alter the ability of stem cells to replicate or form differentiated brain cells. These experiments allowed us to set the time and frequency settings in our separations so that exposure to DEP will have no effect on the cells. The results of our cell sorting experiments demonstrate that specific populations of stem cells, such as those that will form neurons, can be isolated without cell type specific markers using DEP. Furthermore, human embryonic stem cells and NSPCs undergo specific changes as they differentiate that can be detected by DEP. We have found specific cell biophysical properties that reflect the status of stem cells along differentiation pathways. We believe that isolation of NSPCs that specifically generate neurons by our non-toxic and label free procedure will provide purified populations of these cells for basic biological studies, therapeutic approaches, and drug testing.

The main goal of our project is to identify a new method to accurately separate stem cells and their differentiated progeny without the use of cell type-specific markers. The sorting method we are investigating uses dielectrophoresis (DEP), which generates movement of cells toward or away from electrode arrays via non-toxic electric fields. The frequency of the field and the inherent properties of the individual cells provide the basis for separation of unique cell types. Our previous data showed that DEP was capable of distinguishing mouse neural stem/progenitor cells (NSPCs) from more differentiated neural cells (neurons and astrocytes). Furthermore, the dielectric properties of NSPCs that preferentially formed neurons were distinct from those of their astrocyte-biased counterparts, which is important since these NSPC subpopulations cannot currently be discriminated by markers. The specific aims of our project were to determine the applicability of DEP-based separations for neural stem cell populations by generating novel DEP sorting devices and using them to isolate NSPCs on the basis of their fate potential. During the final year of the project, we completed production of a multi-channel device with perpendicular channels controlled by valves. This device proved to be quite robust and was used to sort specific neural cell populations without the use of markers. To determine the applicability of DEP-based separation for stem cells, we have tested whether exposure to DEP alters the cells, sorted cells by DEP frequency and tested their purity, and applied DEP to human NSPCs. Our experiments have shown that DEP exposure does not harm NSPCs when shorter exposures as needed for cell sorting are used. At longer exposure times, particular frequencies damage ~30% of the cells. Importantly, DEP exposure does not alter the ability of stem cells to replicate or form differentiated brain cells. These experiments allowed us to set the time and frequency settings in our separations so that exposure to DEP will have no effect on the cells. The results of our cell sorting experiments demonstrate that distinct populations of stem cells, such as those that will form neurons or those that will form astrocytes, can be isolated without cell type specific markers using DEP. Furthermore, human embryonic stem cells and NSPCs undergo unique changes as they differentiate that can be detected by DEP. We have found particular cell biophysical properties that reflect the status of stem cells along differentiation pathways. We believe that isolation of NSPCs that specifically generate neurons by our non-toxic and label free procedure will provide purified populations of these cells for basic biological studies, therapeutic approaches, and drug testing. Our studies have resulted in 7 publications (4 published or in press, 3 submitted) and the submission of 2 patents.