Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self-renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA-NCAM, which is considered an NP marker. Additionally, cell surface N-linked glycosylation was found to significantly affect cell fate-specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface.
The neural degeneration caused by spinal cord injury leaves a cavity at the injury site that greatly inhibits repair. One approach to promoting repair is to fill the cavity with a scaffold to limit further damage and encourage regrowth. Injectable materials are advantageous scaffolds because they can be placed as a liquid in the lesion site then form a solid in vivo that precisely matches the contours of the lesion. Fibrin is one type of injectable scaffold, but risk of infection from blood borne pathogens has limited its use. We investigated the potential utility of salmon fibrin as an injectable scaffold to treat spinal cord injury since it lacks mammalian infectious agents and encourages greater neuronal extension in vitro than mammalian fibrin or Matrigel®, another injectable material. Female rats received a T9 dorsal hemisection injury and were treated with either salmon or human fibrin at the time of injury while a third group served as untreated controls. Locomotor function was assessed using the BBB scale, bladder function was analyzed by measuring residual urine, and sensory responses were tested by mechanical stimulation (von Frey hairs). Histological analyses quantified the glial scar, lesion volume, and serotonergic fiber density. Rats that received salmon fibrin exhibited significantly improved recovery of both locomotor and bladder function and a greater density of serotonergic innervation caudal to the lesion site without exacerbation of pain. Rats treated with salmon fibrin also exhibited less autophagia than those treated with human fibrin, potentially pointing to amelioration of sensory dysfunction. Glial scar formation and lesion size did not differ significantly among groups. The pattern and timing of salmon fibrin’s effects suggest that it acts on neuronal populations but not by stimulating long tract regeneration. Salmon fibrin clearly has properties distinct from those of mammalian fibrin and is a beneficial injectable scaffold for treatment of spinal cord injury.
The integration of microscale engineering, microfluidics, and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype, function, and physiology. These systems are increasingly being used to assess diverse cell types, such as stem cells, so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example, engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs), which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics, suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs, we investigated whether electric field exposure of varying times affected survival, proliferation, or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival, proliferation, or fate potential revealed by differentiation. Moreover, NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However, lengthy exposure (> 5 min) to frequencies near the crossover frequency (50–100 kHz) led to decreased survival of NSPCs (maximum ~30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension, we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells, which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times, thus providing a basis for developing DEP-based applications for stem cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.